記述言語：英語   掲載種別：研究論文（学術雑誌）  

Colorectal cancer (CRC) exhibits substantial molecular heterogeneity, yet detailed single-cell transcriptomic data from Japanese patients remain limited. Here, we performed single-cell RNA sequencing (scRNA-seq) on tumor tissues and adjacent normal tissues from four Japanese CRC patients to characterize malignant cell populations. We identified a common cancer-associated cell cluster observed across all four cases, characterized by high expression of the long noncoding RNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1). Further analysis revealed a marked decrease in Ras Association Domain Family Member 6 (RASSF6) expression within this cluster, despite clinical datasets reporting that elevated RASSF6 expression correlates with poor overall survival in Western cohorts. Reanalysis of publicly available bulk RNA-seq data from independent East Asian cohorts confirmed that RASSF6 expression was consistently reduced in tumor tissues compared with adjacent normal colon tissues, while KCNQ1OT1 expression showed no apparent differences across cohorts. Collectively, our single-cell analysis delineates a recurrent cancer-associated transcriptional state observed within this cohort and highlights distinct gene expression features that may be underappreciated by bulk or population-averaged analyses, underscoring the value of single-cell profiling for refining molecular interpretations of CRC.

DOI： 10.1111/cas.70335

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Adoptive T cell therapy has shown significant efficacy in cancer treatment, especially in hematologic malignancies, and is increasingly being explored for solid cancers. Combining T cell therapy with conventional treatments holds promise for enhancing therapeutic effects. In this study, we conducted an in vitro inhibitor screening to evaluate the effects of various inhibitors on T cell-mediated cytotoxic activity against cancer cells. Among the candidates, we identified afatinib as an immunosuppressive agent that attenuates T cell cytotoxic activity by reducing interferon-γ (IFN-γ) secretion and suppressing T cell activation. Notably, this IFN-γ reduction was independent of T cell proliferation. RNA-seq analysis revealed that afatinib downregulated the T cell receptor (TCR) pathway signature. RT-qPCR demonstrated a dose-dependent suppression of IFNG mRNA expression in afatinib-treated T cells. Furthermore, afatinib impaired tumor rejection in an immunological memory mouse model that had been previously cured by anti-PD-L1 therapy, suggesting that afatinib may inhibit the function of effector memory T cells. Collectively, our findings highlight afatinib's potential to impair T cell effector functions, indicating that strategic consideration is essential when combining epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), including afatinib, with adoptive T cell therapies.

DOI： 10.1007/s00262-025-04279-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Although various therapeutic options are available for advanced or recurrent non-small cell lung cancer (NSCLC), the optimal criteria for selecting combination therapies involving anti-programmed death-1 (PD-1) or anti-programmed death ligand-1 (PD-L1) antibodies with anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antibodies remain unclear. Clinical trials suggest potential benefits of the treatment with anti-CTLA-4 antibodies in NSCLC with PD-L1 <1%; however, concerns persist regarding the increased risk of immune-related adverse events (irAEs). Therefore, identifying reliable biomarkers to guide the use of anti-CTLA-4 antibodies is crucial. PATIENTS AND METHODS: We performed immunohistochemical staining to assess intratumoral tumor-infiltrating lymphocytes (iTILs) and stromal tumor-infiltrating lymphocytes (sTILs) expressing CD8 or FOXP3 using surgically obtained specimens. The association between these cells and the clinical efficacy of the treatment with nivolumab plus ipilimumab was evaluated using univariate and multivariate analyses in NSCLC patients with PD-L1 <1%. Kaplan-Meier analysis was conducted to assess survival outcomes. RESULTS: Univariate analysis revealed a significant correlation between progression-free survival and sTIL infiltration, but not iTIL infiltration. Patients who responded to therapy exhibited significantly higher CD8+ and lower FOXP3+ iTIL infiltration, as reported previously. Notably, responders also demonstrated significantly higher infiltration of both CD8+ and FOXP3+ sTILs. Moreover, high stromal infiltration of CD8+ T cells was significantly associated with prolonged overall survival, while high FOXP3+ sTILs infiltration showed a trend toward improved overall survival. CONCLUSION: Despite the increased risk of irAEs, patients with high stromal infiltration of CD8+ and FOXP3+ T cells may derive meaningful clinical benefit from anti-CTLA-4 antibody therapy. Assessing these immune parameters could aid in appropriate patient selection and contribute to optimizing therapeutic outcomes.

DOI： 10.21873/anticanres.17944

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Biomarkers that predict durable benefit from immune checkpoint inhibitors (ICIs) and antibody-drug conjugates (ADCs) in metastatic urothelial carcinoma (mUC) remain limited. Tertiary lymphoid structures (TLS), particularly mature TLS (mTLS) characterized by follicular dendritic cell networks and germinal centers, may reflect a highly compartmentalized antitumor immune niche. CASE PRESENTATION: A 77-year-old man with bladder cancer and lung metastases experienced disease progression after two cycles of gemcitabine/cisplatin. Pembrolizumab was initiated but discontinued after a single dose due to destructive thyroiditis; nevertheless, his lung metastases subsequently regressed. Because residual urothelial disease persisted in the bladder, enfortumab vedotin (EV) was initiated. A complete response (CR) was achieved after three cycles and maintained through 10 cycles, at which point treatment was stopped due to fatigue. The patient has remained recurrence-free for over 4 years since the initiation of pembrolizumab. METHODS AND RESULTS: Immunohistochemistry of the pre-treatment transurethral resection of bladder tumor (TUR-Bt) specimen (CD3, CD4, CD8, CD20, CD21, BCL6) revealed numerous TLS adjacent to the tumor, many of which met the criteria for mTLS (CD21+ FDC networks and BCL6+ germinal center B cells). A HALO-based digital pathology spatial analysis demonstrated a highly enriched B- and T-cell microenvironment, with the vast majority of these lymphocytes tightly compartmentalized within the mTLS regions. In an exploratory cohort of six additional sequentially treated mUC cases evaluated using the same methodology, mTLS were not detected, even among cases exhibiting a generalized T-cell infiltration. CONCLUSION: Pre-existing mTLS identified in routine TUR specimens reflect a highly compartmentalized immune niche-distinct from a simple "hot tumor" phenotype-that may help predict which patients are likely to achieve a durable benefit from sequential ICI and ADC therapy, potentially informing treatment selection and sequencing in mUC.

DOI： 10.3389/fonc.2026.1907498

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

T cells recognize peptides presented by human leukocyte antigen molecules on the cell surface, enabling the immune surveillance of pathological conditions such as cancer. Cancer-testis (CT) antigens are promising targets for cancer immunotherapy because of their restricted expression in normal tissues. In this study, we performed antigen screening of T cell receptors isolated from tumor-infiltrating lymphocytes (TILs) in acral melanoma, using cDNA expression cloning and identified a novel CT antigenic epitope encoded by MAGE-A6 with a single nucleotide polymorphism (SNP). This SNP conferred immunogenicity to the epitope, eliciting a robust immune response against tumor cells. While antigen identification has increasingly relied on reverse immunology approaches using reference sequences that do not contain SNPs, forward immunology approaches, such as cDNA expression cloning, directly identify antigens recognized by T cells exhibiting immune responses, enabling the detection of SNP-derived epitopes. Furthermore, in hot tumors such as acral melanoma that are characterized by a low tumor mutational burden, but high TIL infiltration, TILs predominantly respond to shared antigens with high immunogenicity. These findings underscore the utility of forward immunology in antigen discovery and highlight the potential of SNP-dependent tumor antigens in cancer immunotherapy.

DOI： 10.1080/2162402X.2025.2528110

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Placenta accreta spectrum (PAS) is a serious disease leading to complications and maternal death. The objective of the study was to characterize the placental villi and blood vessels of PAS villi histopathologically. We investigated 10 cases of PAS (five cases of placenta increta, two cases of placenta accreta, and three cases of placenta percreta) histologically. Immunohistochemical staining using anti-CD68 or anti-CD163 antibodies was performed to detect and count Hofbauer cells. Immunohistochemical staining with an anti-CD34 antibody was used to detect vascular endothelial cells, and the number and area of vessels were analyzed. The numbers of CD68-positive or CD163-positive Hofbauer cells were larger in PAS cases compared with control cases. The vascular area in villi was smaller in PAS cases compared with control cases. The number of blood vessels in villi was slightly higher in PAS cases than in control cases. The numbers of Hofbauer cells and vessels in villi were larger in PAS cases compared with control cases, whereas the area of vessels in villi was smaller in PAS cases compared with control cases. Although their biological meaning is elusive, these findings provide novel insights into the pathogenesis of PAS, particularly regarding the role of Hofbauer cells in immune-suppressive role and angiogenesis and the alterations in vascular structure and hemodynamics in the chorionic villi.

DOI： 10.1007/s00795-025-00432-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The testis is an immune-privileged organ due to its lack of functional human leukocyte antigen (HLA) class I expression. However, the expression status of HLA class I in testicular seminoma, which is characterized histologically by lymphocytic infiltration, remains unclear. MATERIALS AND METHODS: We performed immunohistochemistry to evaluate HLA class I expression and its spatial relationship with CD8-positive cells in 20 cases of stage I seminoma. Each specimen was scored manually and further analyzed using the HALO image analysis platform. RESULTS: Manual scoring and HALO analysis showed 80 % concordance. HLA class I expression was positive in 13 cases (65 %). Tumors were classified into three categories based on the expression levels. The number of infiltrating CD8-positive cells was significantly correlated with HLA class I expression. Spatial analysis revealed that tumors with higher HLA class I expression had shorter distances between HLA class I-positive tumor cells and CD8-positive cells. CONCLUSION: Seminoma cells expressing HLA class I tended to be located in close proximity to CD8-positive T cells, suggesting a spatial relationship that might influence the tumor immune microenvironment.

DOI： 10.1016/j.prp.2025.156270

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immunomodulatory drugs (IMiDs), such as lenalidomide, are a cornerstone of multiple myeloma (MM) treatment; however, their efficacy remains suboptimal. We previously reported that inhibition of DOT1L, a histone H3 lysine 79 methyltransferase, upregulates interferon (IFN)-regulated genes (IRGs) and exerts anti-MM effects. In this study, we aimed to further elucidate the epigenetic dependency of DOT1L in MM and the mechanism by which its inhibition induces innate immune signaling. Analysis of DepMap portal data revealed that MM cells are preferentially dependent on DOT1L, among epigenetic regulators, for survival. DOT1L inhibition activated type I IFN responses and increased expression of human leukocyte antigen (HLA) class II genes in MM cells. Notably, DOT1L inhibition was associated with induction of DNA damage responses. CRISPR/Cas9-mediated knockout of STING1 attenuated IRG induction and diminished the anti-proliferative effects of DOT1L inhibition, suggesting that activation of STING signaling contributes to its anti-MM activity. Furthermore, DOT1L inhibition downregulated IKZF1/3 and IRF4, which was also associated with IRG induction. Finally, DOT1L inhibition enhanced the anti-MM efficacy of lenalidomide by further upregulating IRGs and suppressing IRF4-MYC signaling. These findings suggest that DOT1L is a preferential epigenetic therapeutic target in MM. Its inhibition not only activates innate immune signaling but also enhances the efficacy of lenalidomide.

DOI： 10.1016/j.canlet.2025.217941

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The development of immunocompetent three-dimensional (3D) culture systems is critical for advancing in vitro models that enable precise analysis of immune-tumor interactions. Here, we report a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues (CAViTs) approach. This system enables spatial compartmentalization of cancer cells and stromal components, including fibroblasts and endothelial cells, thereby mimicking the tumor microenvironment (TME). When co-cultured with tumor-specific cytotoxic T lymphocytes (CTLs), the system permits quantitative analysis of T cell infiltration and cytotoxicity. Moreover, we constructed a cancer-associated fibroblast (CAF)-rich stroma to model immune exclusion. Drug screening using this model identified histone deacetylase (HDAC) inhibitors as agents capable of reducing stromal barrier function by downregulating ECM components, thereby enhancing T cell penetration. This platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma dynamics and accelerating immunotherapeutic discovery. STATEMENT OF SIGNIFICANCE: We present a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues approach. This system enables spatial compartmentalization of cancer cells and stromal components, closely mimicking the tumor microenvironment. Within this model, tumor cell killing by CTLs was successfully observed, resembling a "hot tumor" phenotype. Furthermore, we established a CAF-rich stroma to recapitulate immune exclusion. Drug screening using this platform revealed that HDAC inhibitors enhanced CTL-mediated cytotoxicity. Overall, this platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma interactions and accelerating the discovery of novel immunotherapeutic strategies.

DOI： 10.1016/j.actbio.2025.09.012

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immunoglobulin G4-related disease (IgG4-RD) is a chronic inflammatory condition of unknown etiology characterized by lymphocytic infiltration, fibrosis, and infiltration of IgG4-positive plasma cells. It affects various organs, including the pancreas and salivary glands. Immunological abnormalities are suspected to play a role in its pathogenesis, and there is an epidemiological link to allergic conditions and type 2 inflammation. This study focused on the expression of thymus and activation-regulated chemokine (TARC)/CCL17, which is involved in the migration of T helper 2 and/or regulatory T cells, in salivary gland tissues of patients with IgG4-RD. We analyzed 60 salivary gland biopsy samples obtained from patients at Sapporo Medical University Hospital between 2015 and 2020. Immunohistochemical analysis revealed TARC/CCL17 positivity in 87.2% of histologically confirmed IgG4-RD cases and negativity in 84.6% of histologically unconfirmed but clinically suspected IgG4-RD cases. There was a significant correlation between histologically confirmed IgG4-RD and TARC/CCL17 expression, suggesting its potential diagnostic utility and possible involvement in the pathogenesis of IgG4-RD.

DOI： 10.1080/25785826.2025.2460910

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Despite advancements in the treatment of oral cancer, cancer survival rates remain low, highlighting the need for new therapeutic strategies targeting cancer stem-like cells. Cancer stem-like cells are a small population of cancer cells within tumors that drive recurrence and metastasis. They are often resistant to conventional treatments. Immunotherapy has shown promise against cancer stem-like cells, particularly with the use of cytotoxic T lymphocytes targeting specific markers. Survivin, an apoptosis protein inhibitor, is overexpressed in several malignancies, including oral cancer, and is associated with tumor recurrence and reduced survival. Survivin-2B-specific cytotoxic T lymphocytes were produced and evaluated for their ability to target CD44+ (cancer stem-like cells) and CD44- cells (non-cancer stem-like cells), respectively, from oral cancer cell lines (HSC-2 and HSC-3, respectively). Quantitative polymerase chain reaction (qPCR) analysis confirmed similar survivin-2B expression in both cell types. Cytotoxic T lymphocyte assays revealed the effective lysis of both cancer stem-like cells and CD44- cell populations, supporting the potential of survivin-2B-specific cytotoxic T lymphocytes to overcome cancer stem-like cell-associated resistance. These findings suggest that survivin-2B peptide vaccines are effective in preventing cancer relapse by targeting cancer stem-like cells, with future directions aimed at developing multipeptide "cocktail" vaccines to reduce the risk of immune evasion.

DOI： 10.1111/eos.70019

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In many malignancies, an increased number of tumor-infiltrating lymphocytes (TILs) is recognized as a favorable prognostic factor, with exceptions such as renal cell carcinoma. However, the clinical significance of TIL size remains unclear. T-cell activation by mitogens increases cell size, partly via c-myc expression, suggesting that larger T cells may be more activated. We hypothesized that TIL size might be prognostically relevant in cancer patients. Here, we examined the relationship between the size and number of tumor-infiltrating CD8 + T cells and patient prognosis in 96 cases of esophageal squamous cell carcinoma (ESCC). We employed artificial intelligence (AI) analysis to quantify the mean size of intratumoral CD8+ T cells in each sample. Patients were then divided into "Large" and "Small" CD8+ T cell groups according to the median T-cell size. Similarly, we classified cases into "High" and "Low" groups based on CD8 + T-cell numbers. We found that patients in the Large CD8+ T cell group had significantly better overall survival than those in the Small CD8+ T cell group by a univariate analysis (p = 0.039), but the difference did not reach statistical significance in a multivariate analysis (p = 0.054). Patients in the High CD8 + T cell group had better outcomes than those in the Low CD8+ T cell group. There was no significant correlation between CD8+ T cell size and count, and their combination (Large/High) identified a subgroup of patients with the most favorable prognosis. Our findings suggest that CD8+ T cell size could serve as an independent prognostic marker in ESCC.

DOI： 10.1038/s41598-025-10885-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tumors can evade host immune surveillance by compromising the intracellular antigen processing machinery (APM), such as beta 2 macroglobulin (β2m) or the transporter associated with antigen processing (TAP). Defects in the APM generally result in the downregulation of surface MHC class I (MHC-I) levels. Here, we show that the downregulation of a component of the peptide loading complex (PLC), tapasin, in tumors conversely induces CD8+ T-cell responses and inhibits tumor growth in vivo. Loss of tapasin enhanced the anti-tumor effects of immune checkpoint blockade (ICB) in mouse non-small cell lung and colon cancer models. In contrast to β2m-deficient tumors, the reduced levels of MHC-I in tapasin-deficient tumors were restored by IFN-γ treatment, allowing them to be recognized by CD8+ T cells. These results suggest the presence of a reactive CD8+ T-cell fraction and the ability of immune surveillance to eliminate tumor variants with impaired tapasin expression.

DOI： 10.1111/cas.70027

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The development of effective immunotherapies for solid tumors remains a significant challenge. In previous studies, we identified PVT1, a long non-coding RNA, with the peptide HF10 derived from PVT1, presented by HLA-A24. This study aims to develop a single-chain variable fragment (scFv) that specifically recognizes the HLA-A24/HF10 complex (HF10 scFv) and to evaluate its specificity, reactivity, and therapeutic potential as part of a T cell engaging bispecific antibody (HF10xCD3) in vitro and in vivo. Using a scFv phage display library, we screened for scFv clones targeting the HLA-A24/HF10 peptide complex. The selected HF10 scFv was engineered into an IgG1 format (HF10-hIgG1), which demonstrated high affinity (KD = 2.18 × 10⁻⁸ M) and specific detection of the HLA-A24/HF10 complex on HLA-A24( +)/PVT1( +) tumor cell lines. Furthermore, HF10 scFv was incorporated into a T cell engaging bispecific antibody (HF10xCD3), which induced cytotoxicity in these tumor cell lines. In a mouse xenograft model, HF10xCD3 administration exhibited significant anti-tumor activity. In conclusion, HF10xCD3 represents a promising candidate for immunotherapy targeting solid tumors.

DOI： 10.1007/s00262-025-03976-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Inducing antigen peptide-specific cytotoxic T cells is challenging, partly due to the difficulty of maintaining the quality of antigen-presenting cells, such as dendritic cells. Consequently, artificial antigen-presenting cells (aAPCs) derived from the erythroleukemia cell line K562 have been employed for T cell stimulation. K562-based aAPCs can be utilized for both non-specific and antigen-specific T cell stimulation. Antigen peptide-pulsed aAPCs are commonly used to stimulate T cells with known specific antigenic peptides, which require identifying antigenic peptides from cognate antigen proteins. Therefore, antigen gene-overexpressing aAPCs might be useful for detecting unknown antigenic peptides. In this study, we evaluated the efficacy of cytotoxic T lymphocyte (CTL) induction using antigen gene-overexpressing aAPCs. To enhance antigen presentation efficiency, we assessed the signal peptide (SP) fused with MHC class I trafficking signal (MITD) sequences (SP-MITD). SP-MITD, fused with epitopes from the neoantigen AKF9 and the viral antigen CMVpp65, was transduced into aAPCs. We compared the CTL induction ability of peptide-pulsed aAPCs, only mini-gene-overexpressing aAPCs [SP-MITD (-)], and mini-gene fused with SP-MITD overexpressing aAPCs [SP-MITD (+)]. The SP-MITD (+) gene-overexpressing aAPCs exhibited the highest CTL induction efficiency compared to both peptide-pulsed and SP-MITD (-) gene-overexpressing aAPCs. These findings suggest that antigen gene-fused with SP-MITD transduced aAPCs are highly effective for inducing CTLs specific to both known and unknown antigenic peptides.

DOI： 10.1016/j.bbrep.2025.101946

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tumor-reactive CD4+ T cells often accumulate in the tumor microenvironment (TME) in human cancer, but their functions and roles in antitumor responses remain elusive. Here, we investigated the immunopeptidome of HLA class II-positive (HLA-II+) endometrial cancer with an inflamed TME using a proteogenomic approach. We identified HLA-II neoantigens, one of which induced polyclonal CD4+ tumor-infiltrating lymphocyte responses. We then experimentally demonstrated that neoantigen-specific CD4+ tumor-infiltrating lymphocytes lyse target cells in an HLA-II-dependent manner. Single-cell transcriptomic analysis of the TME coupled with T-cell receptor sequencing revealed the presence of CD4+ T-cell clusters characterized by CXCL13 expression. The CXCL13+ clusters contained two subclusters with distinct cytotoxic gene expression patterns. The identified neoantigen-specific CD4+ T cells were found exclusively in one of the CXCL13+ subclusters characterized by granzyme B and CCL5 expression. These results demonstrate the involvement of tumor-reactive CD4+ T cells with cytotoxic function in immune surveillance of endometrial cancer and reveal their transcriptomic signature.

DOI： 10.1158/2326-6066.CIR-24-0514

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Neoantigen-reactive CD4+ T cells play a key role in the anti-tumor immune response. However, the majority of epithelial tumors are negative for HLA class II (HLA-II) surface expression, and less is known about the processing of HLA-II antigens. Here, we directly identified naturally presented HLA-II neoantigens in HLA-II negative colorectal cancer (CRC) tissue using a proteogenomic approach. The neoantigens were immunogenic and induced patient CD4+ T cells with a Th1-like memory phenotype that produced IFN-γ, IL2 and TNF-α. Multiplex immunohistochemistry (IHC) demonstrated an interaction between Th cells and HLA-II-positive antigen-presenting cells (APCs) at the invasive margin and within the tertiary lymphoid structures (TLS). In our CRC cohort, the density of stromal APCs was associated with HLA-II antigen presentation in the tumor microenvironment (TME), and the number of TLS was positively correlated with the number of somatic mutations in the tumors. These results demonstrate the presence of neoantigen-specific CD4+ surveillance in HLA-II-negative CRC and suggest a potential role for macrophages and dendritic cells (DCs) at the invasive margin and in TLS for antigen presentation. Stromal APCs in the TME can potentially be used as a source for HLA-II neoantigen identification.

DOI： 10.1080/2162402X.2024.2404665

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Adoptive T cell therapy, using T cell receptor-engineered T (TCR-T) cells and chimeric antigen receptor T (CAR-T) cells, is a potent immunotherapy option. Bladder cancer is a prevalent urological malignancy, particularly in cases of muscle invasion and metastasis, for which systemic therapy is crucial. Immunotherapy utilizing immune checkpoint blockade has been approved for bladder cancer treatment. The antitumor effect of an immune checkpoint blockade based on cytotoxic T cells (CTLs) and the patient's immune status is essential. The chemotherapeutic drug cisplatin (CDDP) is a key drug in bladder cancer treatment. However, it has been shown to suppress T cells, making combination therapy with CDDP and immunotherapy difficult. To address this, we developed TCR-T cells specific for bladder cancer cells. In previous studies, we found that the tumor-associated antigen CLSPN is overexpressed in CDDP-resistant bladder cancer cells and that the antigenic peptide HLA-A*02:01/CLSPN1254-1262, encoded by CLSPN, could be targeted by a CTL clone. The TCR was cloned from the HLA-A*02:01/CLSPN1254-1262 specific CTL clone yc3. We also designed a codon-optimized TCR sequence using GeneArt® GeneOptimizer® (Opt TCR) and compared the TCR-T cells using the original TCR sequence (Ori TCR-T cells) and the codon-optimized TCR sequence (Opt TCR-T cells). Opt TCR-T cells exhibited higher TCR transduction efficiency, higher TCR expression levels, higher avidity, and greater cytotoxicity than did Ori TCR-T cells. These results suggest that HLA-A*02:01/CLSPN1254-1262 specific Opt TCR-T cells are promising candidates for CDDP combination therapy.

DOI： 10.1080/21645515.2024.2414542

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Acral lentiginous melanoma (ALM) is the most common melanoma subtype in non-Caucasians. Despite advances in cancer immunotherapy, current immune checkpoint inhibitors remain unsatisfactory for ALM. Hence, we conducted comprehensive immune profiling using single-cell phenotyping with reactivity screening of the T cell receptors of tumor-infiltrating T lymphocytes (TILs) in ALM. Compared with cutaneous melanoma, ALM showed a lower frequency of tumor-reactive CD8 clusters and an enrichment of regulatory T cells with direct tumor recognition ability, suggesting a suppressive immune microenvironment in ALM. Tumor-reactive CD8 TILs showed heterogeneous expression of coinhibitory molecules, including KLRC1 (NKG2A), in subpopulations with therapeutic implications. Overall, our study provides a foundation for enhancing the efficacy of immunotherapy in ALM.

DOI： 10.1126/scitranslmed.adk8832

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Neoantigens arising from somatic mutations are tumor specific and induce antitumor host T cell responses. However, their sequences are individual specific and need to be identified for each patient for therapeutic applications. Here, we present a proteogenomic approach for neoantigen identification, named Neoantigen Selection using a Surrogate Immunopeptidome (NESSIE). This approach uses an autologous wild-type immunopeptidome as a surrogate for the tumor immunopeptidome and allows human leukocyte antigen (HLA)-agnostic identification of both HLA class I (HLA-I) and HLA class II (HLA-II) neoantigens. We demonstrate the direct identification of highly immunogenic HLA-I and HLA-II neoantigens using NESSIE in patients with colorectal cancer and endometrial cancer. Fresh or frozen tumor samples are not required for analysis, making it applicable to many patients in clinical settings. We also demonstrate tumor prevention by vaccination with selected neoantigens in a preclinical mouse model. This approach may benefit personalized T cell-mediated immunotherapies.

DOI： 10.1126/sciadv.ado6491

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Various biomarkers are utilized in the field of breast cancer. Human lymphocyte antigen (HLA) class I molecules have a critical role in cancer immune surveillance. Therefore, this study aimed to assess the HLA class I expression and analyze the correlation with clinicopathologic factors in breast cancer. PATIENTS AND METHODS: We investigated the clinical pathology archives of 150 consecutive patients with breast cancer who underwent a curative operation at the Sapporo Medical University, Japan, from January 2012 to December 2014. Immunohistochemical staining was used to evaluate HLA class I expression and CD8-positive T cell infiltration. The Pearson χ2 test was used to assess HLA class I expression level and clinicopathological parameters. The Kaplan-Meier method was used to evaluate survival and the log-rank test to analyze the differences between survival curves. RESULTS: Patients with dull/negative HLA class I had significantly poor disease-free survival (DFS) compared with those with positive HLA class I (p=0.0073). Univariate analyses revealed that pT, pN, positive lymphatic invasion, and dull/negative HLA class I were significantly associated with DFS. Multivariate analyses revealed dull/negative HLA class I as an independent poor prognostic factor (hazard ratio=2.75, 95% confidence interval=1.30-5.80, p=0.008). CONCLUSION: HLA class I expression level may have a very sensitive prognostic effect on patients with breast cancer.

DOI： 10.21873/anticanres.17233

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: This study evaluated the association between programmed cell death-ligand 1 (PD-L1) and prognosis in patients with cervical cancer treated with postoperative radiation and the impact of neoadjuvant chemotherapy (NAC) on this association. PATIENTS AND METHODS: Immunohistochemical analysis was performed on biopsy specimens from 42 patients who did not receive NAC and from paired samples before (biopsies) and after (resected tissues) chemotherapy from 46 patients who received NAC to determine the association of PD-L1 with radiotherapy outcomes. RESULTS: In the non-NAC group, patients with ≥10% PD-L1-expressing tumor cells prior to treatment had better recurrence-free survival (RFS) than those with <10% PD-L1-expressing tumor cells (p=0.001). In the NAC group, RFS was significantly lower (p=0.005) in the group with a ≥5% reduction of PD-L1 expression in tumor cells after chemotherapy than in those with <5% reduction. In multivariate analysis, only PD-L1 expression (non-NAC group) and the change in PD-L1 expression (NAC group) were associated with RFS. CONCLUSION: Low PD-L1 expression in a cervical tumor prior to treatment was identified as a risk factor for a poor outcome after postoperative radiotherapy. Furthermore, NAC induces an immunological shift that reduces PD-L1 levels in tumor cells, thereby negatively impacting treatment outcomes.

DOI： 10.21873/anticanres.17121

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The recognition by cytotoxic T cells (CTLs) is essential for the clearance of SARS-CoV-2 virus-infected cells. Several viral proteins have been described to be recognized by CTLs. Among them, the spike (S) protein is one of the immunogenic proteins. The S protein acts as a ligand for its receptors, and several mutants with different affinities for its cognate receptors have been reported, and certain mutations in the S protein, such as L452R and Y453F, have been found to inhibit the HLA-A24-restricted CTL response. In this study, we conducted a screening of candidate peptides derived from the S protein, specifically targeting those carrying the HLA-A24 binding motif. Among these peptides, we discovered that NF9 (NYNYLYRLF) represents an immunogenic epitope. CTL clones specific to the NF9 peptide were successfully established. These CTL clones exhibited the ability to recognize endogenously expressed NF9 peptide. Interestingly, the CTL clone demonstrated cross-reactivity with the Y453F peptide (NYNYLFRLF) but not with the L452R peptide (NYNYRYRLF). The CTL clone was able to identify the endogenously expressed Y453F mutant peptide. These findings imply that the NF9-specific CTL clone possesses the capability to recognize and respond to the Y453F mutant peptide.

DOI： 10.1080/25785826.2024.2304363

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Epidermal keratinocytes, forming the outermost layer of the human body, serve as a crucial barrier against diverse external stressors such as ultraviolet radiation. Proper keratinocyte differentiation and effective responses to external stimuli are pivotal for maintaining barrier integrity. Heat is one such stimulus that triggers the synthesis of heat shock proteins (HSPs) when cells are exposed to temperatures above 42 °C. Additionally, activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) occurs at 42 °C. Here, we explore the interplay between TRPV1 signaling and HSP induction in human keratinocytes. Both heat and capsaicin, a TRPV1 agonist, induce expression of HSP27, HSP70, and HSP90 in keratinocytes. Interestingly, pharmacological inhibition of TRPV1 attenuates heat-induced HSP27 expression, but not that of HSP70 or HSP90. Furthermore, both heat and capsaicin stimulation result in distinct phosphorylation patterns of heat shock factor 1 (HSF1), with phosphorylation at serine 326 being a common feature. Notably, genetic manipulation to mimic dephosphorylation of HSF1 at serine 326 reduces HSP27 levels. Additionally, ΔNp63, a key regulator of epidermal differentiation, negatively modulates HSP27 expression independently of HSF1 phosphorylation status. While heat stimulation has no effect on ΔNp63 expression, capsaicin reduces its levels. The precise role of TRPV1 signaling in keratinocytes warrants further investigation for a comprehensive understanding of its impact on barrier function.

DOI： 10.1016/j.bbrc.2024.149817

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The application of deep learning algorithms to predict the molecular profiles of various cancers from digital images of hematoxylin and eosin (H&E)-stained slides has been reported in recent years, mainly for gastric and colon cancers. In this study, we investigated the potential use of H&E-stained endometrial cancer slide images to predict the associated mismatch repair (MMR) status. H&E-stained slide images were collected from 127 cases of the primary lesion of endometrial cancer. After digitization using a Nanozoomer virtual slide scanner (Hamamatsu Photonics), we segmented the scanned images into 5397 tiles of 512 × 512 pixels. The MMR proteins (PMS2, MSH6) were immunohistochemically stained, classified into MMR proficient/deficient, and annotated for each case and tile. We trained several neural networks, including convolutional and attention-based networks, using tiles annotated with the MMR status. Among the tested networks, ResNet50 exhibited the highest area under the receiver operating characteristic curve (AUROC) of 0.91 for predicting the MMR status. The constructed prediction algorithm may be applicable to other molecular profiles and useful for pre-screening before implementing other, more costly genetic profiling tests.

DOI： 10.3390/cancers16101810

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immunotherapy using immune checkpoint inhibitors (ICIs) has shown superior efficacy compared with conventional chemotherapy in certain cancer types, establishing immunotherapy as the fourth standard treatment alongside surgical intervention, chemotherapy, and radiotherapy. In cancer immunotherapy employing ICIs, CD8-positive cytotoxic T lymphocytes are recognized as the primary effector cells. For effective clinical outcomes, it is essential that the targeted cancer cells express HLA class I molecules to present antigenic peptides derived from the tumor. However, cancer cells utilize various mechanisms to downregulate or lose HLA class I molecules from their surface, resulting in evasion from immune surveillance. Correlations between prognosis and the integrity of HLA class I molecules expressed by cancer cells have been consistently found across different types of cancer. This paper provides an overview of the regulatory mechanisms of HLA class I molecules and their role in cancer immunotherapy, with a particular emphasis on the significance of utilizing pathological tissues to evaluate HLA class I molecules expressed in cancer cells.

DOI： 10.1111/tan.15472

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. MATERIALS AND METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. CONCLUSION: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

DOI： 10.21873/anticanres.16989

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記述言語：英語  

DOI： 10.1111/1346-8138.17070

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記述言語：英語  

DOI： 10.1111/1346-8138.17043

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The immune microenvironment in cancer correlates with cancer progression and patient prognosis. Cancer immune microenvironment evaluation, based on CD3+ and CD8+ T cell infiltration at the center and invasive margin of the tumor, is defined as the immunoscore. An international multicenter analysis revealed that the immunoscore can accurately predict the prognosis of patients with colorectal cancer (CRC) (stage I, II, and III). However, no markers are currently available to predict the prognosis in patients with stage IV CRC. We thus aimed to analyze the immune microenvironment in patients with stage IV CRC in this study. PATIENTS AND METHODS: We analyzed the immune microenvironment of patients with stage IV CRC using immunohistochemical (IHC) staining. We evaluated the expressions of CD8 and the cases were divided into CD8 high (CD8Hi) and CD8 low (CD8Low) groups according to median CD8 expression. HLA class 1 (HLA1) expression was also evaluated using IHC staining and the cases were divided into HLA1Hi group and HLA1Low group according to 50% of HLA1 expression rate. CD8×HLA1 score was defined by the combination of CD8 and HLA1 expressions. RESULTS: CD8Hi and HLA1Hi cases were associated with better prognosis compared with CD8Low and HLA1Low cases according to a log-rank test, respectively. We defined a novel biomarker by combining CD8+ T-cell infiltration and HLA1 expression, referred to as the CD8×HLA1 score. We found that CD8×HLA1Hi cases predicted patient prognosis better than CD8×HLA1Int and CD8×HLA1Low according to a log-rank test. CONCLUSION: The combination of CD8+ T cell infiltration and HLA1 expression is crucial for cancer immune microenvironment evaluation in CRCs.

DOI： 10.21873/anticanres.16958

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: Although immune checkpoint inhibitors (ICIs), together with cytotoxic chemotherapy (chemoimmunotherapy), have been adapted for the initial treatment of extensive-disease small-cell lung cancer (ED-SCLC), they have achieved limited success. In ED-SCLC, a subtype of SCLC, the expression of immune-related molecules and clinical data are not well understood in relation to ICI treatment efficiency. METHODS: We examined lung biopsy specimens from patients diagnosed with ED-SCLC treated with chemoimmunotherapy or chemotherapy. SCLC subtype, expression of HLA class I, and infiltration of CD8-positive cells were examined using immunohistochemistry (IHC). Subsequently, the association between clinical factors, IHC results, and progression-free survival or overall survival was assessed. RESULTS: Most of the cases showed the achaete-scute homolog 1 (ASCL1) subtype. Among the 75 SCLC cases, 29 expressed high levels of HLA class I, while 46 showed low levels or a negative result; 33 patients were characterized as CD8-high, whereas 42 were CD8-low. In the chemoimmunotherapy cohort, multivariate analysis revealed a correlation between CD8-high and improved survival. Specifically, patients in the CD8-high group of the chemoimmunotherapy cohort experienced enhanced survival compared to those in the chemotherapy cohort, which was attributed to ICI addition. IHC subtype analysis demonstrated a survival advantage in the SCLC-I and SCLC-A groups when ICI was combined with chemotherapy compared to chemotherapy alone. CONCLUSION: Our study highlights the predictive value of IHC-classified subtypes and CD8-positive cell infiltration in estimating outcomes for patients with ED-SCLC treated with chemoimmunotherapy as a first-line therapy. These findings have practical implications for daily clinical assessments and treatment decisions.

DOI： 10.1007/s00432-024-05652-2

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Early diagnosis is essential for the safer perinatal management of placenta accreta spectrum (PAS). We used transcriptome analysis to investigate diagnostic maternal serum biomarkers and the mechanisms of PAS development. We analyzed eight formalin-fixed paraffin-embedded placental specimens from two placenta increta and three placenta percreta cases who underwent cesarean hysterectomy at Sapporo Medical University Hospital between 2013 and 2019. Invaded placental regions were isolated from the uterine myometrium and RNA was extracted. The transcriptome difference between normal placenta and PAS was analyzed by microarray analysis. The PAS group showed markedly decreased expression of placenta-specific genes such as LGALS13 and the pregnancy-specific beta-1-glycoprotein (PSG) family. Term enrichment analysis revealed changes in genes related to cellular protein catabolic process, female pregnancy, autophagy, and metabolism of lipids. From the highly dysregulated genes in the PAS group, we investigated the expression of PSG family members, which are secreted into the intervillous space and can be detected in maternal serum from the early stage of pregnancy. The gene expression level of PSG6 in particular was progressively decreased from placenta increta to percreta. The PSG family, especially PSG6, is a potential biomarker for PAS diagnosis.

DOI： 10.1007/s00795-023-00371-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and β constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.

DOI： 10.1111/cas.15967

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations. MATERIALS AND METHODS: Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells. RESULTS: JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay. CONCLUSION: Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.

DOI： 10.21873/cgp.20460

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Automated measurement of immunostained samples can enable more convenient and objective prediction of treatment outcome from radiotherapy. We aimed to validate the performance of the QuPath image analysis software in immune cell markers detection by comparing QuPath cell counting results with those of physician manual cell counting. PATIENTS AND METHODS: CD8- and FoxP3-stained cervical, CD8-stained oropharyngeal, and Ku70-stained prostate cancer tumor sections were analyzed in 104 cervical, 92 oropharyngeal, and 58 prostate cancer patients undergoing radiotherapy at our Institution. RESULTS: QuPath and manual counts were highly correlated. When divided into two groups using ROC curves, the agreement between QuPath and manual counts was 89.4% for CD8 and 88.5% for FoxP3 in cervical cancer, 87.0% for CD8 in oropharyngeal cancer and 80.7% for Ku70 in prostate cancer. In cervical cancer, the high CD8 group based on QuPath counts had a better prognosis and the low CD8 group had a significantly worse prognosis [p=0.0003; 5-year overall survival (OS), 65.9% vs. 34.7%]. QuPath counts were more predictive than manual counts. Similar results were observed for FoxP3 in cervical cancer (p=0.002; 5-year OS, 62.1% vs. 33.6%) and CD8 in oropharyngeal cancer (p=0.013; 5-year OS, 80.2% vs. 47.2%). In prostate cancer, high Ku70 group had worse and low group significantly better outcome [p=0.007; 10-year progression-free survival (PFS), 56.0% vs. 93.8%]. CONCLUSION: QuPath showed a strong correlation with manual counting, confirming its utility and accuracy and potential applicability in clinical practice.

DOI： 10.21873/invivo.13593

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Radiotherapy (RT) for advanced oropharyngeal cancer (OPC) is effective, especially when combined with chemotherapy (CRT). However, its success can vary depending on factors, such as tumor stage, HPV infection (p16 status), and the patient's nutritional and immune status. This study examined the controlling nutritional status (CONUT) score and tumor immunity as predictive factors for treatment outcomes in OPC, aiming to develop a personalized risk score. PATIENTS AND METHODS: A retrospective analysis was conducted on 84 patients with OPC treated with definitive RT or CRT, and survival outcomes were compared based on various factors, including BMI, CONUT score, CD8 expression, and HLA class II expression. RESULTS: We observed better overall survival (OS) rates in CD8-positive patients and those with higher HLA class II expression. The univariate analysis identified stage, p16 status, BMI, CONUT score, and CD8 expression as significantly associated with OS. In multivariate analysis, stage, BMI, and CONUT score remained significant predictors of OS. A risk scoring system was developed based on stage, p16 status, BMI, CONUT score, and CD8 expression. Patients were categorized into low-risk and high-risk groups, with significantly better survival in the low-risk group. CONCLUSION: A combined risk score incorporating clinical, nutritional, and immune factors can improve the prediction of treatment outcomes for OPC patients. This risk stratification may enable personalized treatment plans and improve ΟS rates.

DOI： 10.21873/cdp.10397

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Preoperative intra-arterial chemoradiotherapy (IACRT) can improve the outcome and reduce the extent of surgery in patients with advanced oral cancer. However, the response to this regimen varies among patients, which may be related to the immune status of the tumor. We investigated the effects of proteins involved in tumor immunity on the outcomes of combined IACRT and surgery for oral cancer. We examined CD8 + and FoxP3 + tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression on immune cells and tumor cells in pretreatment biopsy samples from 69 patients diagnosed with oral cancer treated with IACRT at our institution during 2000-2020. Patients with abundant CD8 + TILs had significantly better 5-year disease-specific survival (DSS) compared to that of patients with less infiltration of these cells (P = 0.016). Patients with higher FoxP3 + T-cells invasion had significantly better DSS compared to that of less FoxP3 (P = 0.005). Patients with high PD-L1 expression in tumor cells and immune cells had significantly better DSS than that of patients with low PD-L1 expression in these cells (P = 0.009 and P = 0.025, respectively). Collectively, these results suggest that the tumor immune microenvironment could affect outcomes of IACRT treatment in oral cancer.

DOI： 10.1007/s00795-023-00367-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Eribulin inhibits microtubule polymerization and improves the overall survival of patients with recurrent metastatic breast cancer. A subgroup analysis revealed a low neutrophil to lymphocyte ratio (NLR) (<3) to be a prognostic factor of eribulin treatment. We thus hypothesized that eribulin might be related to the immune response for breast cancer cells and we analyzed the effects of eribulin on the immune system. Immunohistochemical staining revealed that human leukocyte antigen (HLA) class I expression was increased in clinical samples after eribulin treatment. In vitro assays revealed that eribulin treatment increased HLA class I expression in breast cancer line cells. RNA-sequencing demonstrated that eribulin treatment increased the expression of the NOD-like family CARD domain-containing 5 (NLRC5), a master regulator of HLA class I expression. Eribulin treatment increased the NY-ESO-1-specific T-cell receptor (TCR) transduced T (TCR-T) cell response for New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) overexpressed breast cancer cells. The eribulin and TCR-T combined therapy model revealed that eribulin and immunotherapy using TCR-T cells has a synergistic effect. In summary, eribulin increases the expression of HLA class 1 via HLA class 1 transactivatior NLRC5 and eribulin combination with immunotherapy can be effective for the treatment of breast cancer.

DOI： 10.1111/cas.15986

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The mortality rate of oral cancer has not improved over the past three decades despite remarkable advances in cancer therapies. Oral cancers contain a subpopulation of cancer stem cells (CSCs) that share characteristics associated with normal stem cells, including self-renewal and multi-differentiation potential. CSCs are tumorigenic, play a critical role in cancer infiltration, recurrence, and distant metastasis, and significantly contribute to drug resistance to current therapeutic strategies, including immunotherapy. Cytotoxic CD8+ T lymphocytes (CTLs) are key immune cells that effectively recognize peptide antigens presented by the major histocompatibility complex class I molecules. Increasing evidence suggests that cancer antigen-specific targeting by CTLs effectively regulates CSCs that drive cancer progression. In this study, we utilized data from public domains and performed various bioassays on human oral squamous cell carcinoma clinical samples and cell lines, including HSC-2 and HSC-3, to investigate the potential role of olfactory receptor family 7 subfamily C member 1 (OR7C1), a seven transmembrane G-protein-coupled olfactory receptor that is also expressed in nonolfactory tissues and was previously reported as a novel marker and target of colon cancer initiating cell-targeted immunotherapy, in CSC-targeted treatment against oral cancer. We found that the OR7C1 gene was expressed only in oral CSCs, and that CTLs reacted with human leukocyte antigen-A24-restricted OR7C1 oral CSC-specific peptides. Taken together, our findings suggest that OR7C1 represents a novel target for potent CSC-targeted immunotherapy in oral cancer.

DOI： 10.1111/cas.15873

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The current coronavirus disease 2019 (COVID-19) pandemic that is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a significant threat to public health. Although vaccines based on the mRNA of the SARS-CoV-2 spike protein have been developed to induce both cellular and humoral immunity against SARS-CoV-2, there have been some concerns raised about their high cost, particularly in developing countries. In the present study, we aim to identify an immunogenic peptide in the SARS-CoV-2 spike protein to activate cellular immunity, particularly CD4+ helper T lymphocytes (Th cells), which are a commander of immune system. SARS-CoV-2 spike protein-derived peptides Spike448-477 and Spike489-513(N501Y)-specific CD4+ Th cell lines were generated by repetitive stimulation of healthy donor-derived CD4+T-cells with each peptide. Their HLA-restrictions were addressed by using blocking antibodies against HLA and HLA-transfected L-cells. The epitopes of Spike448-477-specific CD4+ Th cell lines were defined using a series of 7-14-mer overlapping truncated peptides and alanine-substituted epitope peptides. To address responsiveness of these CD4+ Th cell lines to several SARS-CoV-2 variants, we stimulated the CD4+ Th cell lines with mutated peptides. We addressed whether these identified peptides were useful for monitoring T-cell-based immune responses in vaccinated donors using the IFN-γ ELISpot assay. The Spike448-477 peptide was found to be a promiscuous peptide presented by HLA- DRB1*08:02, DR53, and DPB1*02:02. Although HLA-DPB1*02:02-restricted CD4+ Th cells did not response to some peptides with the L452R and L452Q mutations, the other CD4+ Th cells were not affected by any mutant peptides. We developed two tetramers to detect HLA-DRB1*08:02/Spike449-463- and Spike449-463(L452R/Y453F)-recognizing CD4+ Th cells. Spike489-513(N501Y) peptide was also a promiscuously presented to HLA-DRB1*09:01 and DRB1*15:02. The T-cell responses specific to both peptides Spike448-477 and Spike489-513 were detected in PBMCs after vaccinations. In addition, we observed that the Spike448-477 peptide activated both CD8+ T-cells and CD4+ Th cells in individuals receiving mRNA vaccines. SARS-CoV-2 spike protein-derived peptides, Spike448-477 and Spike489-513, include several epitopes that are presented by multiple HLA-class II alleles to activate CD4+ Th cells, which are considered useful for monitoring the establishment of acquired immunity after vaccination.

DOI： 10.1016/j.heliyon.2023.e20192

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Immunotherapy using immune checkpoint inhibitors (ICIs) has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC). Although several ICI options are available, the treatment regimen for NSCLC with large size tumors (large NSCLC) is controversial and the efficacy of anti-CTLA-4 antibody is unclear. This study thus investigated potential biomarkers for CTLA-4 blockade. PATIENTS AND METHODS: The correlation between tumor diameter and treatment duration was examined in patients with advanced NSCLC treated with anti-PD-1 antibody monotherapy in our institution. In addition, the ratio of tumor-infiltrating CD8+ T cells and regulatory T (Treg) cells in small and large size NSCLC was also evaluated using immunohistochemical staining. Finally, the efficacy of treatment with anti-CTLA-4 antibody against large NSCLC was investigated. RESULTS: A negative correlation was found between tumor diameter and treatment duration in patients treated with anti-PD-1 antibody monotherapy. Immuno-histochemical staining revealed that Treg cell infiltration was significantly higher in large NSCLC tumors than in small tumors. Among the patients with large NSCLC, the ICI regimen including anti-CTLA-4 antibody showed significant efficacies. CONCLUSION: Anti-PD-1 antibody monotherapy might be less effective against large NSCLC due to the infiltration of Treg cells. Therefore, it might be appropriate for large NSCLC to select a treatment including an anti-CTLA-4 antibody, which can target Treg cells.

DOI： 10.21873/anticanres.16606

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously showed that upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in vascular endothelial cells promotes tumor angiogenesis. In the present study, we aimed to clarify the role of stromal AEBP1/ACLP expression in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis showed that ACLP is abundantly expressed in cancer-associated fibroblasts (CAFs) in primary OSCC tissues and that upregulated expression of ACLP is associated with disease progression. Analysis using CAFs obtained from surgically resected OSCCs showed that the expression of AEBP1/ACLP in CAFs is upregulated by co-culture with OSCC cells or treatment with TGF-β1, suggesting cancer-cell-derived TGF-β1 induces AEBP1/ACLP in CAFs. Collagen gel contraction assays showed that ACLP contributes to the activation of CAFs. In addition, CAF-derived ACLP promotes migration, invasion, and in vivo tumor formation by OSCC cells. Notably, tumor stromal ACLP expression correlated positively with collagen expression and correlated inversely with CD8+ T cell infiltration into primary OSCC tumors. Boyden chamber assays suggested that ACLP in CAFs may attenuate CD8+ T cell migration. Our results suggest that stromal ACLP contributes to the development of OSCCs, and that ACLP is a potential therapeutic target.

DOI： 10.3390/cancers15174303

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS: An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS: Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS: The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes.

DOI： 10.3390/cancers15164045

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Evasion from immunity is a major obstacle to the achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion are theoretically associated with tumor heterogeneity and progression by conferring novel properties on tumor cells, including drug resistance and metastatic capacity; however, their impact on immune evasion remains unknown. Here, we investigated the potency of tumor-macrophage hybrids in immune evasion. Hybrids were established by co-culture of a melanoma cell line (A375 cells) and type 2 macrophages. The hybrids showed greater migration ability and greater tumorigenicity than the parental melanoma cells. The hybrids showed heterogeneous sensitivity to New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T-cell receptor-transduced T (TCR-T) cells and two out of four hybrid clones showed less sensitivity to TCR-T compared with the parental cells. An in vitro tumor heterogeneity model revealed that the TCR-T cells preferentially killed the parental cells compared with the hybrids and the survival rate of the hybrids was higher than that of the parental cells, indicating that the hybrids evade killing by TCR-T cells efficiently. Analysis of a single-cell RNA sequencing dataset of patients with melanoma revealed that a few macrophages expressed RNA encoding melanoma differentiation antigens including melan A, tyrosinase, and premelanosome protein, which indicated the presence of hybrids in primary melanoma. In addition, the number of potential hybrids was correlated with a poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. © 2023 The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.6083

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

DOI： 10.1007/s00262-023-03388-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitors (ICIs) for various types of malignancy, including non-small-cell lung cancer, have improved prognosis in some cases. Granuloma formation after ICI administration suggests a tumor antigen-specific cytotoxic T cell response with abundant interferon-gamma production, which can be used to estimate the curative effect of ICIs. In this report, we present a case with a resected lung lesion, clinically suspected to be lung cancer, that consisted of a granulomatous lesion. A tumor was also found in the duodenum that was presumed to be derived from the pulmonary pleomorphic carcinoma. Duodenal tumor cells highly expressed PD-L1, suggesting PD-1/PD-L1 axis-mediated immune escape. As expected, pembrolizumab induced a complete response for the duodenal lesion. Interestingly, in histopathological analysis, the duodenal lesion was also replaced by an epithelial granuloma and multinucleated giant cells. We conclude that autoimmunity regressed the untreated primary lung lesion spontaneously, while the metastatic duodenal lesion responded to PD-1 blockade. Tumor-associated epithelioid granulomas, even before ICI administration, may be an important pathological finding indicating an immune response with interferon-gamma production by cytotoxic T cells to the tumor.

DOI： 10.1080/25785826.2023.2193283

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Since its discovery in late 2019, severe acute respiratory syndrome coronavirus 2 has spread around the world, causing millions of deaths due to coronavirus disease 2019 (COVID-19). Numerous clinical and post-mortem investigations of COVID-19 cases have found myriad clinical and pathological manifestations of the disease. In this report, we present three autopsy cases in which, despite weaning from extracorporeal membrane oxygenation (ECMO), extensive intestinal epithelial shedding, probably due to ischemia, was followed by massive watery diarrhea and the spread of infection via the portal vein due to bacterial translocation, which resulted in cholangitis lenta. Thrombophilia was attributed to ECMO usage and COVID-19-related vascular endothelial damage. These cases provide instructive findings showing that the loss of the intestinal barrier may be the underlying cause of severe watery diarrhea and liver failure in COVID-19 patients, especially with ECMO usage.

DOI： 10.1093/omcr/omad031

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Malignant melanoma is a fatal skin cancer and is among the most immunogenic malignancies expressing melanoma-differentiation antigens and neoantigens. SRY-related HMG-box 10 (SOX10) is a transcription factor and a neural-crest differentiation marker that is used as a diagnostic marker for melanoma whilst playing a role in melanoma initiation through activation of the SOX10-MITF axis. SOX10 was shown to play a role in melanoma initiation by inducing expression of immune checkpoint molecules (e.g., HVEM and CEACAM1). In this study, we aimed to investigate the relationship between SOX10 and the expression an immune checkpoint molecule, programmed death-1 ligand 1 (PD-L1). MATERIALS AND METHODS: SOX10 overexpression and knockdown was performed using SOX10 gene transfection and SOX10 siRNA transfection into A375 melanoma cells. PD-L1 expression was assessed by flow cytometry and western blotting. T cell response was evaluated using NY-ESO-1 specific TCR-transduced T (TCR-T) cells by IFNγ ELISPOT assay. RESULTS: SOX10 overexpression increased the expression of PD-L1, whereas SOX10 knockdown, using siRNA, decreased its expression. IFNγ ELISPOT assay revealed that overexpression of SOX10 decreased the susceptibility of cells to NY-ESO-1-specific TCR-T cells. CONCLUSION: SOX10 has a role in the intrinsic immune suppressive mechanisms of melanoma through expression of PD-L1.

DOI： 10.21873/anticanres.16296

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: As therapy for solid tumours, various tumour antigens have been selected as targets, but CAR-T cells targeting these antigens have shown limited efficacy, in contrast to the effectiveness of CAR-T cells targeting haematological malignancies. In a previous report, we identified a cancer-testis antigen, DNAJB8. DNAJB8 plays a major role in tumorigenicity in cancer stem-like cells/cancer-initiating cells (CSCs/CICs). Here, we report a DNAJB8-reactive CAR yielding anti-tumour effects against renal cell carcinoma (RCC) and osteosarcoma. METHODS: We constructed a second-generation chimeric antigen receptor (CAR) against HLA-A*24:02/DNAJB8-derived peptide (DNAJB_143) complex (B10 CAR). The reactivity of B10-CAR T cells against T2-A24 cells pulsed with the cognate peptide and an RCC and osteosarcoma cell lines were quantified. The effects of adoptive cell transfer (ACT) therapy were assessed using in vivo xenografted mice models. RESULTS: B10 CAR-T cells recognised DNAJB8_143-pulsed T2-A24 cells and HLA-A*24:02(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. Moreover, ACT using B10 CAR-T cells showed anti-tumour effects against RCC and osteosarcoma cells. CONCLUSION: B10 CAR-T cells could show specific cytotoxicity against RCC and osteosarcoma cells in vitro and in vivo. B10 CAR-T cells targeting the CSC/CIC antigen DNAJB8 might be a candidate immunotherapy for carcinoma and sarcoma.

DOI： 10.1038/s41416-022-02100-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Resistance of cervical cancer to radiotherapy with concurrent chemotherapy (CCRT) results in a poor prognosis. To identify new biomarkers for predicting the treatment response and prognosis, we explored exosomal microRNA (miRNA) expression signatures associated with the outcome of cervical cancer patients treated with CCRT. Exosomes were isolated from the plasma of 45 patients prior to CCRT during 2014-2020, and miRNA analysis was performed by next-generation sequencing. At a median follow-up of 38 months, 26 patients were recurrence free, 15 patients had died of the disease, and 4 patients received salvage chemotherapy due to distant metastasis. Of the 2522 miRNAs detected, 9 (miR-148a-5p, 1915-3p, 3960, 183-5p, 196b-5p, 200c-3p, 182-5p, 374a-5p, and 431-5p) showed differential expression between the recurrence-free and recurrence groups. Patients were divided into high- and low-risk groups according to the cutoff of the miRNAs-based risk score calculated from respective expression levels. The high-risk group had significantly worse disease-specific survival than the low-risk group (p < 0.001). In addition, miR-374a-5p and miR-431-5p expression showed a weak inverse correlation with tumor-infiltrating CD8+ and FOXP3+ T cells, suggesting a potential inhibitory effect on CCRT by suppressing tumor immunity. This miRNA signature could improve non-invasive monitoring and personalized treatment for cervical cancer.

DOI： 10.1007/s00795-022-00338-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Arachidonate 5-lipoxygenase (ALOX5) is a cardinal enzyme in the synthesis of leukotrienes, which are powerful immune-regulating lipid mediators. We previously reported that ALOX5 is preferentially expressed in B lymphocytes in the mantle zone of human lymphoid tissue. In the context of physiological relevance, the loss of the Alox5 gene in mice significantly impairs the development of follicular B helper T cells and antibody production. However, ALOX5 expression in B-cell lymphomas has not been investigated in detail. In this study, we examined ALOX5 expression in representative B-cell lymphomas and non-neoplastic lymphoid tissues by immunohistochemistry with a commercially available anti-ALOX5 antibody that can be used on formalin-fixed paraffin-embedded specimens. Interestingly, 22/22 cases of mantle cell lymphoma, 7/7 cases of chronic lymphocytic leukemia/small cell lymphoma, and 20/20 cases of follicular lymphoma expressed ALOX5. A small proportion of extranodal marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma or nodal marginal zone lymphoma cases were positive for ALOX5 (2/13 or 1/3, respectively). In contrast, no cases with diffuse large B-cell lymphoma, regardless of germinal center B cell (GCB) or non-GCB type, expressed ALOX5 (0/25 cases). These findings suggest that ALOX5 may be a novel marker for identifying the cell of origin of B-cell lymphoma. Further investigation is required to clarify the biological significance of ALOX5 expression in lymphoma cells.

DOI： 10.1016/j.prp.2023.154328

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.

DOI： 10.1002/cam4.5310

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

DOI： 10.3390/ijms24010771

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pulmonary pleomorphic carcinoma is rare among lung tumors. Pulmonary pleomorphic carcinoma is resistant to chemotherapy. However, treatment with taxane anticancer agents and immune checkpoint inhibitors has been reported to be effective. When using immune checkpoint inhibitors, pseudoprogression and true progression are difficult to distinguish, and immune-related adverse events (irAEs) are common. We herein report a patient with simultaneous pseudoprogression and irAEs after combined therapy with cytotoxic agents and an immune checkpoint inhibitor for pulmonary pleomorphic carcinoma. Immune checkpoint inhibitors are effective against pulmonary pleomorphic carcinoma, but patients should be monitored for pseudoprogression and irAEs.

DOI： 10.2169/internalmedicine.8916-21

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: In this study, we evaluated the prognostic value of Immunoscore in patients with stage I−III colon cancer (CC) in the Asian population. These patients were originally included in an international study led by the Society for Immunotherapy of Cancer (SITC) on 2681 patients with AJCC/UICC-TNM stages I−III CC. METHODS: CD3+ and cytotoxic CD8+ T-lymphocyte densities were quantified in the tumor and invasive margin by digital pathology. The association of Immunoscore with prognosis was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS). RESULTS: Immunoscore stratified Asian patients (n = 423) into different risk categories and was not impacted by age. Recurrence-free rates at 3 years were 78.5%, 85.2%, and 98.3% for a Low, Intermediate, and High Immunoscore, respectively (HR[Low-vs-High] = 7.26 (95% CI 1.75−30.19); p = 0.0064). A High Immunoscore showed a significant association with prolonged TTR, OS, and DFS (p < 0.05). In Cox multivariable analysis stratified by center, Immunoscore association with TTR was independent (HR[Low-vs-Int+High] = 2.22 (95% CI 1.10−4.55) p = 0.0269) of the patient’s gender, T-stage, N-stage, sidedness, and MSI status. A significant association of a High Immunoscore with prolonged TTR was also found among MSS (HR[Low-vs-Int+High] = 4.58 (95% CI 2.27−9.23); p ≤ 0.0001), stage II (HR[Low-vs-Int+High] = 2.72 (95% CI 1.35−5.51); p = 0.0052), low-risk stage-II (HR[Low-vs-Int+High] = 2.62 (95% CI 1.21−5.68); p = 0.0146), and high-risk stage II patients (HR[Low-vs-Int+High] = 3.11 (95% CI 1.39−6.91); p = 0.0055). CONCLUSION: A High Immunoscore is significantly associated with the prolonged survival of CC patients within the Asian population.

DOI： 10.3390/cancers14184346

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We developed an immunohistogram representing an individual cancer-immunity cycle based on immunohistochemical analyses. We evaluated its ability to predict the efficacy of immune checkpoint inhibitors (ICI) in 11 patients with urothelial carcinoma and 7 patients with renal cell carcinoma who underwent surgery and received ICIs for disease recurrence. Immunohistochemical analyses for CD8, TIA-1, HLA class I, HLA-DR, and PD-L1 were performed and scored 0-3. T-cell infiltration pattern was classified into desert, excluded, partially inflamed, and inflamed. Tumors with an inflamed or partially inflamed pattern and positive scores (score ≥ 1) for all five immune markers were classified as "immune-hot" and others as "immune-cold." Association between the immunohistogram and ICI treatment efficacy was evaluated with objective response rate, disease control rate (DCR), progression-free survival (PFS), and cancer-specific survival (CSS). Eight (44%) and 10 (56%) patients had immune-hot and immune-cold tumors, respectively. Immune-hot tumors showed a higher DCR (100% vs. 40%, p < 0.01), longer PFS (median unreached for hot, 1.3 months for cold, p < 0.01), and longer CSS (median unreached for hot, 3.3 months for cold, p < 0.01) than immune-cold tumors. The immunohistogram could be clinically useful as an accessible biomarker for precision cancer immunotherapy in urological cancer.

DOI： 10.1038/s41598-022-14700-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

CD8+ T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8+ T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.

DOI： 10.4049/jimmunol.2100717

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Carriers of oncogenic human T-cell leukemia virus type 1 (HTLV-1) can develop adult T-cell leukemia/lymphoma (ATLL). While an increasing number of animal models of HTLV-1 infection have revealed that malignant tumors with a histiocytic phenotype can arise, they have not been reported in humans. Here, we present a 79-year-old female HTLV-1 carrier who presented with a swollen lymph node. Histological examination revealed that the lymph node was replaced with a malignant spindle cell tumor, but not ATLL. Immunohistochemical analysis indicated that the tumor was positive for histiocytic (CD68 and CD163) and myogenic (α-smooth muscle actin, desmin, and caldesmon) markers, suggesting some differential diagnoses. We could not reach a definitive diagnosis under the current notion of the disease entity. In addition, we could not provide an exact causal relationship between HTLV-1 infection and the development of the current tumor. Nevertheless, this tumor may be a human counterpart of murine HTLV-1-related histiocytic tumors. Curiously, the tumor showed a good response to chemotherapy with the combination of cyclophosphamide, vincristine, and prednisone, a standard approach for ATLL. This case might represent a novel entity of an HTLV-1-related malignant tumor. Further accumulation of case reports will certainly contribute to our understanding of human HTLV-1-related disease and the mechanism of viral oncogenesis.

DOI： 10.1016/j.prp.2022.153935

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Despite the considerable success of cancer immunotherapy with immune checkpoint inhibitors, their nonspecific release of the immunosuppressive mechanism is often associated with immune-related adverse events (irAEs). irAEs significantly disturb patients' quality of life and can even be life-threatening. Therefore, the appropriate management of irAEs is crucial for the development of further reliable cancer immunotherapies. irAEs have the appearance of ordinary autoimmune diseases in one aspect but often have distinct features. Although the detailed pathogenesis of irAEs remains unclear, increasing numbers of studies have provided numerous clues. Here, we review the current knowledge on irAEs, particularly from an immunopathological basis.

DOI： 10.1080/25785826.2021.1976942

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human papillomavirus (HPV)-related cancer is one of the diseases entities for which the applications of radiotherapy have been increasing. Recently, the process of carcinogenesis from HPV infection and the mechanism of tumor immunity that develops during disease progression have been elucidated. In this review, we will describe the mechanism of tumor immunity and how chemoradiotherapy may overcome and improve the efficacy of tumor immunity. We will also discuss the usefulness of proteins involved with tumor immunity as a predictive marker of radiotherapy response, and present an overview of ongoing clinical trials of combinations of immune checkpoint inhibitors and radiotherapy to demonstrate the promising combination therapy that has been currently emerging.

DOI： 10.1007/s11604-021-01231-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDHhigh clone cells were established. ALDHhigh clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDHhigh clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDHhigh clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

DOI： 10.1007/s00262-021-03025-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitors (ICIs) are used in cancer immunotherapy to block programmed death-1 and cytotoxic T-lymphocyte antigen 4, but the response rate for ICIs is still low and tumor cell heterogeneity is considered to be responsible for resistance to immunotherapy. Tumor-infiltrating lymphocytes (TILs) have an essential role in the anti-tumor effect of cancer immunotherapy; however, the specificity of TILs in renal cell carcinoma (RCC) is elusive. In this study, we analyzed a 58-year-old case with clear cell RCC (ccRCC) with the tumor showing macroscopic and microscopic heterogeneity. The tumor was composed of low-grade and high-grade ccRCC. A tumor cell line (1226 RCC cells) and TILs were isolated from the high-grade ccRCC lesion, and a TIL clone recognized a novel neoantigen peptide (YVVPGSPCL) encoded by a missense mutation of the tensin 1 (TNS1) gene in a human leukocyte antigen-C*03:03-restricted fashion. The TNS1 gene mutation was not detected in the low-grade ccRCC lesion and the TIL clone did not recognized low-grade ccRCC cells. The missense mutation of TNS1 encoding the S1309Y mutation was found to be related to cell migration by gene over-expression. These findings suggest that macroscopically and microscopically heterogenous tumors might show heterogenous gene mutations and reactivity to TILs.

DOI： 10.1007/s00262-021-03048-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The association between type 2 diabetes mellitus and prostate cancer is still under investigation, and the relationship between hyperinsulinemia and prostate cancer stem-like cells (CSCs) is elusive. Here, we investigated the function of insulin/AKT signaling in prostate CSCs. We isolated prostate CSCs as aldehyde dehydrogenase 1-high (ALDH1high) cells from the human prostate cancer 22Rv1 cell line using an ALDEFLUOR assay and established several ALDH1high and ALDH1low clones. ALDH1high clones showed high ALDH1 expression which is a putative CSC marker; however, they showed heterogeneity regarding tumorigenicity and resistance to radiation and chemotherapy. Interestingly, all ALDH1high clones showed lower phosphorylated AKT (Ser473) (pAKT) levels than the ALDH1low clones. PI3K/AKT signaling is a key cell survival pathway and we analyzed radiation resistance under AKT signaling activation by insulin. Insulin increased pAKT levels in ALDH1high and ALDH1low cells; the fold increase rate of pAKT was higher in ALDH1high cells than in ALDH1low cells. Insulin induced resistance to radiation and chemotherapy in ALDH1high cells, and the increased levels of pAKT induced by insulin were significantly related to radiation resistance. These results suggest that ALDH1 suppresses baseline pAKT levels, but AKT can be activated by insulin, leading to treatment resistance.

DOI： 10.1016/j.bbrc.2021.12.095

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Although many cervical cytology diagnostic support systems have been developed, it is challenging to classify overlapping cell clusters with a variety of patterns in the same way that humans do. In this study, we developed a fast and accurate system for the detection and classification of atypical cell clusters by using a two-step algorithm based on two different deep learning algorithms. METHODS: We created 919 cell images from liquid-based cervical cytological samples collected at Sapporo Medical University and annotated them based on the Bethesda system as a dataset for machine learning. Most of the images captured overlapping and crowded cells, and images were oversampled by digital processing. The detection system consists of two steps: (1) detection of atypical cells using You Only Look Once v4 (YOLOv4) and (2) classification of the detected cells using ResNeSt. A label smoothing algorithm was used for the dataset in the second classification step. This method annotates multiple correct classes from a single cell image with a smooth probability distribution. RESULTS: The first step, cell detection by YOLOv4, was able to detect all atypical cells above ASC-US without any observed false negatives. The detected cell images were then analyzed in the second step, cell classification by the ResNeSt algorithm, which exhibited average accuracy and F-measure values of 90.5% and 70.5%, respectively. The oversampling of the training image and label smoothing algorithm contributed to the improvement of the system's accuracy. CONCLUSION: This system combines two deep learning algorithms to enable accurate detection and classification of cell clusters based on the Bethesda system, which has been difficult to achieve in the past. We will conduct further research and development of this system as a platform for augmented reality microscopes for cytological diagnosis.

DOI： 10.1002/cam4.4460

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

YTH domain-containing 2 (YTHDC2) is known to be an important regulator for RNA metabolism. Here, we show that YTHDC2 is essential for breast cancer tumorigenesis and metastasis. We examined YTHDC2 expression levels by immunohistochemistry in human breast tumor tissues from 99 patients and found a significantly positive correlation between the YTHDC2 expression level and the tumor stage. We established YTHDC2-knocked-down cell lines using four breast cancer cell lines with different subtypes. Knockdown of YTHDC2 attenuated the sphere-forming and the metastatic ability of breast cancer cells. Although stemness and EMT markers, such as SOX2, c-MYC, and NANOG, were downregulated in several YTHDC2-knocked-down breast cancer cells, a common target gene of YTHDC2 in breast cancer cells was not identified. These findings suggest that while YTHDC2 is involved in malignant progression of breast cancers, the mechanism by which YTHDC2 regulates those phenotypes is different between subtypes of breast cancers.

DOI： 10.1155/2022/9188920

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

CD8+ T cells recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathologic conditions such as cancer. Advances in proteogenomic analysis of HLA ligandomes have demonstrated that cells present a subset of cryptic peptides derived from noncoding regions of the genome; however, the roles of cryptic HLA ligands in tumor immunity remain unknown. In the current study, we comprehensively and quantitatively investigated the HLA class I ligandome of a set of human colorectal cancer and matched normal tissues, showing that cryptic translation products accounted for approximately 5% of the HLA class I ligandome. We also found that a peptide encoded by the long noncoding RNA (lncRNA) PVT1 was predominantly enriched in multiple colorectal cancer tissues. The PVT1 gene is located downstream of the MYC gene in the genome and is aberrantly overexpressed across a variety of cancers, reflecting its oncogenic property. The PVT1 peptide was recognized by patient CD8+ tumor-infiltrating lymphocytes, as well as peripheral blood mononuclear cells, suggesting the presence of patient immune surveillance. Our findings show that peptides can be translated from lncRNAs and presented by HLA class I and that cancer patient T cells are capable of sensing aberrations in noncoding regions of the genome.

DOI： 10.1158/2326-6066.CIR-20-0964

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

To supplement clinical decision-making in the management of cervical cancer, various prognostic factors, including tumor immune microenvironments, were examined in patients with cervical cancer treated with definitive chemoradiotherapy. We retrospectively analyzed the expression of CD8, FoxP3, HLA-1, PD-L1, and XRCC4 in 100 cases of cervical cancer. The observed tumor immune microenvironments were also classified into three types: inflamed, excluded, and cold type. Less FoxP3+ T cells and cold-type tumor were found to be poor prognostic factors in addition to non-SCC, large pre-treatment tumor volume, and three or less cycles of concurrent chemotherapy based on multivariate analysis. Cold-type tumors had significantly worse prognoses than the other two types, whereas inflamed- and excluded-type tumors showed similar 5-year disease-specific survival (P < 0.001; 0% vs. 60.3% vs. 72.3%). Radiotherapy could overcome the inhibitory immune microenvironment that occurs in excluded type. Individualized combination therapy adapted to pre-treatment tumor immunity may be necessary to improve radiotherapy outcomes in cervical cancer.

DOI： 10.1007/s00795-021-00290-w

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Barrier disruption and an excessive immune response in keratinocytes are now considered to have important roles in the pathophysiology of atopic dermatitis (AD). Furthermore, disturbed keratinocyte differentiation is considered to underlie AD. ΔNp63, a p53-like transcription factor, is a major regulator of keratinocyte differentiation. However, the functional significance of ΔNp63 in AD has not been clarified. OBJECTIVE: In this study, we aimed to investigate the influence of the type 2 inflammatory environment on ΔNp63 expression and AD-associated molecules regulated by ΔNp63 in keratinocytes. METHODS: The immunohistochemical expression profiles of ΔNp63 and AD-related molecules were evaluated in human skin tissue. The function of ΔNp63 in the regulation of AD-related molecules and the influence of the type 2 inflammatory environment on ΔNp63 expression were investigated using human primary keratinocytes. Expression of ΔNp63 was manipulated using the RNA interfering method. RESULTS: In healthy skin tissue, we observed an inverse expression pattern between ∆Np63 and some barrier-related proteins including filaggrin, caspase-14, claudin-1, and claudin-4. ΔNp63 regulated expression of these genes and proteins. In addition, production of IL-1β and IL-33, pro-inflammatory cytokines, was modulated by ΔNp63. Furthermore, prolonged IL-13 exposure increased the thickness of the three-dimensional culture of keratinocytes. IL-13 interfered with ΔNp63 downregulation during calcium-induced keratinocyte differentiation. IL-13 modulated some barrier-related and inflammation-related molecules, which were regulated by ΔNp63. CONCLUSIONS: We have shown that ΔNp63 modulated AD-related barrier and inflammatory molecules. In addition, ΔNp63 expression was affected by IL-4/IL-13. IL-13-ΔNp63 axis would integrate two major factors of AD pathogenesis: dysregulated barrier and inflammation.

DOI： 10.1002/iid3.427

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry-based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch repair-deficient CRC (dMMR-CRC) tissue sample carrying 3967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of nonmutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.

DOI： 10.1172/jci.insight.146356

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The immune system is often called a double-edged sword, due to the inextricable link between cancer immunity and allergy/autoimmunity. Intriguingly, a growing number of cases have been reported in which PD-1 blockade triggers the exacerbation of tuberculosis (TB), an organ-invasive granulomatous disease caused by bacterial infection. As a result, the exacerbation of TB is now considered a severe adverse effect of nivolumab and pembrolizumab. In this letter, we report the strong expression of PD-L1 in epithelioid granulomatous lesions in tuberculosis, sarcoidosis, Crohn's disease, and foreign body granuloma. In addition, we discussed the exacerbation of tuberculosis after anti-PD-1 antibody-based cancer immunotherapy.

DOI： 10.1080/21645515.2020.1870364

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs), including nivolumab and pembrolizumab, have recently been shown to have clinical benefits in patients with advanced non-small cell lung cancer (NSCLC). The novel tumour responses to these agents are changing the management of patients with cancer. Pseudo-progression of disease (pseudo-PD), that is, an initial flare followed by shrinkage of the tumour, has been described as a distinctive response to ICIs. However, pseudo-PD manifest initial progression and is difficult to segregate with hyper progressive disease (HPD). We, therefore, analysed a case with pseudo-PD histologically. PATIENTS AND METHODS: A 68-year-old Japanese man with stage IV non-small cell lung carcinoma (NSCLC) was treated by anti-PD-1 antibody (pembrolizumab). Four weeks later after second time treatment with pembrolizumab, the patient showed severe melena followed by Trousseau syndrome and died at day 174 after first treatment by pembrolizumab, suggesting HPD clinically. Primary lesion and metastatic lesions were analysed histologically. RESULTS: Histological analysis revealed that NSCLC cells expressed PD-L1, and CD8+ tumor-infiltrated lymphocytes (TILs) were observed. CD8+ TILs showed higher rates of PD-1 indicating that lesions were of the inflamed type and the case was pseudo-PD. Furthermore, it was found that cancer cells expressed MUC1. CONCLUSION: The clinical appearance of the case was aggressive after treatment by pembrolizumab, and the case seemed to be HPD; however, histological analysis revealed that the case was likely pseudo-PD. Therefore, careful histological evaluation is important when investigating the clinical response to an ICI and mucin expression might be a predictive marker for Trousseau syndrome.

DOI： 10.21873/anticanres.15161

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Effective management of immune-related adverse events in patients receiving immunotherapy for cancer is problematic. In this report, we present the case of a 58-year-old man with advanced clear cell renal cell carcinoma who responded well to a combination of ipilimumab and nivolumab. However, after two courses of treatment, he developed fulminant hepatitis and died. An autopsy confirmed that the primary lesion in the left kidney was more than 99% necrotic with only six small residual tumor lesions. These lesions were infiltrated by large numbers of CD8-positive/TIA-1-positive lymphocytes. However, a metastatic lesion in the right kidney harbored few lymphocytes. Furthermore, the tumor cells in the metastatic lesion and one of the residual lesions showed decreased expression of HLA class I molecules, which are a prerequisite for cytotoxic T-lymphocyte-mediated immunotherapy in tumor cells. In this patient, more than 80% of hepatocytes were destroyed and the parenchyma was infiltrated with CD8-positive/TIA-1-positive lymphocytes. The patient had polyuria, which was attributed to neurohypophysitis caused by the infiltration of CD8-positive/TIA-1-positive lymphocytes. We believe that this is an instructive case for immuno-oncologists.

DOI： 10.1080/25785826.2020.1788229

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously, we investigated gene expression in a high aldehyde dehydrogenase 1 expression (ALDH1high) population of urothelial carcinoma (UC) cells as UC cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) and found that NRG1 expression was upregulated in ALDH1high cells. NRG1 is a trophic factor that contains an epidermal growth factor (EGF)-like domain that signals by stimulating ERBB receptor tyrosine kinases and the cytoplasmic domain. NRG1 has been determined to be involved in frequent gene fusions with other partners in several malignancies and has a role in carcinogenesis through the NRG1 EGF-like domain and its cognitive receptor ERBBs. We thus aimed to elucidate the function of NRG1 in UC CSCs/CICs in this study. Both NRG1α and NRG1-β1 were preferentially expressed in ALDH1high cells compared with ALDH1low cells; however, siRNA experiments revealed that NRG1-β1 but not NRG1-α has a role in sphere formation. The EGF-like domain of NRG1 had a role in sphere formation of UC cells to some extent but was not essential. The intracellular domain of NRG1 did not have a role in sphere-formation. Inhibition of γ-secretase suppressed sphere formation. These findings indicate that cleavage of NRG1-β1 by γ-secretase plays an important role in UC CSC/CIC proliferation; however, the downstream targets of NRG1-β1 remain elusive.

DOI： 10.1016/j.bbrc.2021.03.038

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitors (ICIs) have provided an additional treatment option for various types of human cancers. However, ICIs often induce various immune-related adverse events (irAEs). Enterocolitis is a major irAE with poorly understood histopathological characteristics. In this study, we retrospectively investigated the histopathology of colon tissue samples from 17 patients treated with ICIs. There were two major histological patterns of colitis: an ulcerative colitis-like pattern and a graft vs host disease-like pattern. Although these two patterns of colitis were mutually exclusive, both patterns often showed a characteristic that we call "subepithelial surface granulomatosis" (SSG), which has not been reported in other types of colitis. SSG was found even in colon tissue without symptoms or endoscopic findings of colitis. Given the increasing reports of sarcoid reaction or exacerbation of tuberculosis after treatment with ICIs, granuloma formation could be a histological hallmark of systemic immune activation by ICIs. Although statistical significance was not obtained, probably because of the small sample size, SSG may be a surrogate biomarker of systemic anticancer immune activation. We propose that a prospective study with larger sample size be performed.

DOI： 10.1111/cas.14773

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mismatch repair protein deficiency (dMMR) is a favorable prognostic factor in colorectal cancer. It is also associated with aberrant expression of HLA class I molecules, which are required for cytotoxic T lymphocyte-mediated cancer immunotherapy. Because dMMR is frequently also found in endometrial cancers (ECs), we retrospectively investigated the expression of mismatch repair proteins and HLA class I molecules in 127 EC patients. In this study, EC patients being treated in our hospital were recruited from 2005 to 2009 and observed until December 2017. Lesion specimens were evaluated via immunohistochemistry for MSH6 and PMS2 (mismatch repair proteins) and HLA class I molecules. Expression of these molecules was statistically related to clinical and pathological factors and prognosis. dMMR was detected in 33 patients and did not correlate with the expression level of HLA class I molecules (P = 0.60). On the other hand, unexpectedly, multivariate analysis revealed that intact expression of HLA class I molecules was associated with p53 overexpression (P = 0.004). Neither dMMR nor decreased expression of HLA class I molecules were prognostic factors. These results are inconsistent with previous findings for colorectal cancer. A distinctive local tissue immune microenvironment would underlie the discrepancy in the results between EC and colorectal cancer.

DOI： 10.1007/s00795-020-00254-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co-localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin- and caveolae-dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K-Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin- and caveolae-dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K-Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.

DOI： 10.1111/cas.14735

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

A 72-year-old man was diagnosed with tumors outside of the stomach and mesentery of the small intestine on abdominal computed tomography. Histopathological examination of an endoscopic ultrasound-guided fine-needle aspiration biopsy specimen confirmed the diagnosis of lymph node metastasis of a neuroendocrine tumor (NET). Gastroscopy, colonoscopy, small bowel capsule endoscopy, somatostatin receptor scintigraphy, and 18F-fluorodeoxyglucose positron emission tomography were performed. However, the primary lesion could not be diagnosed. The patient underwent surgery, and an ileal submucosal tumor, which was not identified preoperatively in addition to the aforementioned abdominal tumors, was detected. All tumors were diagnosed as NET, and the ileal tumor was considered the primary lesion. The patient has shown no recurrence postoperatively. The current study presents a case of an ileal NET with lymph node metastases in a patient in whom the primary lesion remained preoperatively undiagnosed.

DOI： 10.11405/nisshoshi.118.943

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Extensive research over 100 years has demonstrated that tumors can be eliminated by the autologous immune system. Without doubt, immunotherapy is now a standard treatment along with surgery, chemotherapy, and radiotherapy; however, the field of cancer immunotherapy is continuing to develop. The current challenges for the use of immunotherapy are to enhance its clinical efficacy, reduce side effects, and develop predictive biomarkers. Given that histopathological analysis provides molecular and morphological information on humans in vivo, its importance will continue to grow. This review article outlines the basic knowledge that is essential for the research and daily practice of immune checkpoint inhibitor-based cancer immunotherapy from the perspective of histopathology.

DOI： 10.3389/fonc.2021.679095

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

INTRODUCTION: Immune checkpoint inhibitors are now a standard therapeutic option for lung adenocarcinoma. However, Immune checkpoint inhibitors often induce various immune-related adverse events. CASE PRESENTATION: The patient was a 78-year-old woman with lung adenocarcinoma who had a partial response to pembrolizumab. During treatment, she complained of pollakiuria and nocturia with painful micturition. Histological analysis revealed infiltration of CD8-positive and/or TIA-1 cytotoxic granule-associated RNA binding protein-positive lymphocytes and programmed death-ligand 1 expression in the urothelium. A diagnosis of immune-related adverse event cystitis was made based on these clinical and pathological findings. The patient's subjective symptoms and findings on cystoscopy improved dramatically after treatment with prednisolone. CONCLUSION: Immune checkpoint inhibitors-induced cystitis is extremely rare. This report is the first to include an immunohistochemical analysis of the urothelial epithelium in immune-related adverse event cystitis and describes an instructive case.

DOI： 10.1002/iju5.12211

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVE: To evaluate proteins related to tumor immune response and treatment outcome from radiotherapy for uterine cervical cancer patients. METHODS: We performed a retrospective immunohistochemical staining of 81 patients with uterine cervical cancer who underwent definitive radiotherapy. We examined the expression of programmed death ligand 1, human leukocyte antigen class I, tumor-infiltrating CD8+, and forkhead box P3+ (FoxP3+) T cells in tumor tissues. RESULTS: In biopsy specimen, patients with a higher number of CD8+ T cells and FoxP3+ T cells had a better disease-specific survival than patients with a lower number of CD8+ T cells and FoxP3+ cells (P = 0.018 and P = 0.009). Multivariate analysis showed that equivalent dose in 2 Gy fractions (EQD2) of the minimum dose to 90% of the high-risk clinical target volume, FoxP3+ T cells and expression of human leukocyte antigen class I were significant prognostic factors. When the EQD2 is 70 Gy or more, a higher local control rate is obtained regardless of the number of CD8- or FoxP3-positive cells. When EQD2 is <70 Gy, the number of CD8-positive cells has a significant impact on treatment outcome: the recurrence rate (local recurrence rate + distant metastasis rate) was 46.2% in the group with a CD8 value of 230 or higher, whereas the recurrence rate was 75.7% in the group with a CD8 value of less than 230. CONCLUSION: The combination of CD8 or FoxP3 with EQD2 can be potentially useful to predict the treatment results of radiotherapy for cervical cancer, leading to individualized optimal selection of treatment for cervical cancer.

DOI： 10.1093/jjco/hyaa149

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

DOI： 10.1038/s41416-020-1017-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The tumor microenvironment (TME) balances tumor growth and suppression through humoral factors and cell-cell interactions. In oral squamous cell carcinoma (OSCC), TMEs have been associated with prognosis of cancer patients and are evaluated by microscopy; however, these methods of evaluation vary among studies. MATERIALS AND METHODS: To evaluate the TME, borderline microenvironment fibrosis (bMF) was evaluated histologically in 236 OSCC cases and used to determine the clinicopathological status. RESULTS: bMF was observed in 47% (110 in 236 cases) of OSCC cases and associated with higher T category, N category, stage, histological grade and mode of invasion. bMF-positive was related to overall survival (OS) and progression-free survival (PFS). Multivariate analysis revealed that bMF-positive was an independent factor for OS in all cases [n=226; HR=1.683 (1.018-2.781); p=0.042], especially in T1+T2 cases [n=186; HR=1.926 (1.079-3.440); p=0.024], and PFS in all cases [n=226; HR=2.254 (1.397-3.637); p=0.001]. CONCLUSION: bMF may act as a novel biomarker for OSCC.

DOI： 10.21873/anticanres.14434

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: To evaluate radiotherapy-induced changes in the expression of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), and human leukocyte antigen class I (HLA-1) in patients with uterine cervical cancer, as well as infiltration of CD8+ and Forkhead box P3+ (FoxP3+) T lymphocytes into tumor tissue and the prognostic value of these parameters. MATERIALS AND METHODS: We performed immunohistochemical analysis of pre-radiotherapy biopsies and corresponding post-radiotherapy resected tissues in 104 uterine cervical cancer patients undergoing preoperative chemoradiotherapy or radiotherapy alone. We scored the expression of various proteins to distinguish positive from negative samples. RESULTS: PD-L1-expressing tumor cells (PD-L1 TC) increased significantly after chemoradiotherapy (p = 0.043). CD8+ T cell infiltration (p = 0.002) and FoxP3+ T cell infiltration (p = 0.003) decreased significantly after chemoradiotherapy. Expression of PD‑1, PD-L1-expressing immune cells (PD-L1 IC), and HLA‑1 did not change after chemoradiotherapy. In biopsy specimens obtained before chemoradiotherapy or radiotherapy, greater infiltration of CD8+ T cells (p = 0.001) and FoxP3+ T cells (p = 0.003) were significant predictors of better overall survival (OS). In surgical specimens obtained after chemoradiotherapy or radiotherapy, greater infiltration of PD-L1 TC was the only significant predictor of better OS (p < 0.001) and was related to a significantly lower probability of out-of-field recurrence (p = 0.005). CONCLUSION: Chemoradiotherapy induced an immunologic shift that increased PD-L1 TC. Chemoradiotherapy has immunological effects that can influence the results of treatment for uterine cervical cancer.

DOI： 10.1007/s00066-019-01571-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent studies have revealed that metabolic reprogramming is closely associated with epithelial-mesenchymal transition (EMT) during cancer progression. Aldolase A (ALDOA) is a key glycolytic enzyme that is highly expressed in several types of cancer. In this study, we found that ALDOA is highly expressed in uterine cervical adenocarcinoma and that high ALDOA expression promotes EMT to increase malignant potentials, such as metastasis and invasiveness, in cervical adenocarcinoma cells. In human surgical specimens, ALDOA was highly expressed in cervical adenocarcinoma and high ALDOA expression was correlated with lymph node metastasis, lymphovascular infiltration, and short overall survival. Suppression of ALDOA expression significantly reduced cell growth, migration, and invasiveness of cervical cancer cells. Aldolase A expression was partially regulated by hypoxia-inducible factor-1α (HIF-1α). Shotgun proteome analysis revealed that cell-cell adhesion-related proteins were significantly increased in ALDOA-overexpressing cells. Interestingly, overexpression of ALDOA caused severe morphological changes, including a cuboidal-to-spindle shape shift and reduced microvilli formation, coincident with modulation of the expression of typical EMT-related proteins. Overexpression of ALDOA increased migration and invasion in vitro. Furthermore, overexpression of ALDOA induced HIF-1α, suggesting a positive feedback loop between ALDOA and HIF-1α. In conclusion, ALDOA is overexpressed in cervical adenocarcinoma and contributes to malignant potentials of tumor cells through modulation of HIF-1α signaling. The feedback loop between ALDOA and HIF-1α could become a therapeutic target to improve the prognosis of this malignancy.

DOI： 10.1111/cas.14524

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The case of a patient with metastatic renal cell carcinoma who exhibited the abscopal effect following treatment by anti-programmed death-1 (PD-1) antibody is presented. A 40-year-old woman was diagnosed with an 8.2-cm renal tumor without distant metastases, and radical nephrectomy was subsequently performed. Pathological examination revealed a clear cell renal cell carcinoma. At 3 months after surgery, the patient developed one lung metastasis. Following treatment with interferon and three types of tyrosine kinase inhibitors, anti-PD1 antibody (nivolumab) was started. During the treatment, para-aortic/supraclavicular lymph nodes and several lung lesions remained, although other lesions decreased markedly. The patient was subsequently treated by palliative radiotherapy to the para-aortic and supraclavicular lymph nodes for pain control. After the radiotherapy, the lung lesions previously refractory to nivolumab started to decrease, probably due to an abscopal effect. Additionally, the laboratory data and Karnofsky Performance Status improved. Histological re-examination of the primary lesion revealed heterogeneity of the immunological microenvironment, which may be associated with the heterogeneity of treatment sensitivity.

DOI： 10.3892/etm.2020.8423

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Peptide-based immunotherapy does not usually elicit strong immunological and clinical responses in patients with end-stage cancer, including sarcoma. Here we report a myxofibrosarcoma patient who showed a strong clinical response to peptide vaccinations and whose immune responses were reboosted by anti-PD1 therapy combined with peptide vaccinations. The 46-year-old man showed a strong response to the peptide vaccinations (papillomavirus binding factor peptide, survivin-2B peptide, incomplete Freund's adjuvant, and polyethylene glycol-conjugated interferon-alpha 2a) and subsequent wide necrosis and massive infiltration of CD8+ T cells in a recurrent tumor. The patient's immune responses weakened after surgical resection; however, they were reboosted following the administration of nivolumab combined with peptide vaccinations. Thus, anti-PD1 therapy combined with peptide vaccinations might be beneficial, as suggested by the observations in this sarcoma patient.

DOI： 10.1007/s00262-019-02455-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.

DOI： 10.1111/cas.14229

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2-step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN-2B) plus interferon-β (IFNβ); (ii) SVN-2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN-2B plus IFNβ. The primary endpoint was progression-free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty-three patients were randomly assigned to receive SVN-2B plus IFNβ (n = 30), SVN-2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B-specific CTLs were found to be increased in the SVN-2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN-2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN-2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN-2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

DOI： 10.1111/cas.14106

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN-2B), a 9-mer antigenic peptide encoded by survivin, and an SVN-2B peptide vaccine-based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN-2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN-2B peptide vaccination and 6 who had not, as negative controls. The expression of immune-related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme-linked immunospot assay. Histological analysis revealed dense infiltration of CD8+ T cells in some lesions in patients who had received the SVN-2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN-2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor-specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.

DOI： 10.1111/cas.14099

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

DOI： 10.4049/jimmunol.1900029

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Immune checkpoint inhibitors(ICIs)have provided great success in cancer treatment field, and immunotherapies using ICIs have become standard therapy for several cancers. Cancer stem-like cells(CSCs)are defined by their higher tumorigenicity and resistance to chemotherapy and radiotherapy, thus they are supposed to be responsible for recurrence and distant metastasis. Therefore, control of CSCs is a key factor to improve patients' prognosis. In this review article, we summarize the expression of PD-L1, a molecular target of ICIs, in CSCs, and discuss the possibility of CSC-targeting immunotherapy using ICIs.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Immune checkpoint inhibitors (ICIs) have provided more options in the treatment of lung cancer. However, ICIs can cause several unfavorable reactions generally referred to as immune-related adverse effects. CASE PRESENTATION: In this report, we present the case of a 52-year-old woman with successful regression of pleomorphic carcinoma of the lung following nivolumab therapy. She developed purpura fulminans (PF) ultimately resulting in amputation of both lower extremities. Blood tests revealed thrombocytopenia with increased serum soluble IL-2 receptor, ferritin, and triglyceride levels suggesting hemophagocytic lymphohistiocytosis (HLH). In addition, serum A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 activity was decreased, suggesting thrombotic thrombocytopenic purpura (TTP). Further detailed analysis revealed severe hypercytokinemia including increased levels of IL-1β, IL-6, IL-10, TNFα, IFNγ, and G-CSF. CONCLUSION: The severe systemic inflammatory reaction and impaired peripheral circulation in this patient was attributed to excessive immunological effect induced by nivolumab resulting in cytokine release syndrome (CRS). This is the first report of a patient with multiple pathological conditions including HLH, TTP-like condition, and PF presumably arising from ICI-induced CRS. Further accumulating thoroughly investigated cases would lead to better understanding of the disease and development of reliable cancer immunotherapy.

DOI： 10.1186/s40425-019-0582-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Uterine endometrial carcinoma is one of the common cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are tumorigenic and are resistant to treatments, thus they are focused as treatment targets. However, the heterogeneity of CSCs/CICs is still elusive, and we therefore analyzed CSCs/CICs at the clonal level. We previously established sphere-cultured CSCs/CICs from primary human uterine endometrial carcinoma, and we isolated several clones from CSCs/CICs in this study. Interestingly, we established two types of clones based on the growth pattern. The clones were termed sphere clones (S clones) and leukemia-like clones (LL clones). Functional analysis revealed that S clones are resistant to chemotherapy, whereas LL clones are sensitive to chemotherapy. On the other hand, S clones are less tumorigenic, while LL clones are highly tumorigenic. Transcriptome analysis using serial analysis of gene expression sequencing (SAGE-Seq) revealed distinctive gene expression profiles in S clone cells and LL clone cells. The results indicate that CSCs/CICs are composed of functionally heterogenic subpopulations including highly tumorigenic clones and treatment-resistant clones and that the characteristics of CSCs/CICs might be determined by the characteristics of different clones that compose CSCs/CICs.

DOI： 10.1016/j.yexmp.2018.11.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bronchial epithelial cells serve as a physical barrier at the forefront of the immune system. Barrier disruption and an excessive immune response of the bronchial epithelium contribute to the pathophysiology of asthma, a chronic bronchial inflammatory disease. The purpose of this study was to investigate the functional significance of ΔNp63, a p53-like transcription factor expressed by the basal bronchial epithelium. The immunohistochemical expression profile of ΔNp63 was evaluated in human bronchial tissue derived from asthma patients. The role of ΔNp63 in apoptosis inhibition and production of soluble mediators was investigated in vitro with cultured BEAS-2B bronchial epithelial cells using molecular biological analysis. In healthy bronchial tissue, ΔNp63-positive basal epithelial cells were covered with differentiated ΔNp63-negative cells but in the asthmatic airway, ΔNp63-positive cells were directly exposed to the bronchial lumen due to severe epithelial shedding. ΔNp63 regulated bronchial apoptosis in response to Toll-like receptor 3 stimulation. On the other hand, expression of ΔNp63 was modulated by stimulation with trypsin and SLIGKV, protease-activated receptor 2 ligands. Further phenotypic analysis revealed that ΔNp63 controlled the transcriptional expression and protein release of some epithelium-derived proinflammatory cytokines and endogenous protease inhibitors. We conclude that ΔNp63 modulates the bronchial epithelial response to viral infection. At the same time, ΔNp63 expression is influenced by proteases, which are abundant in house dust mites. Therefore, the ΔNp63 axis would be intimately involved in these two major triggers of asthma exacerbations, viral infection and protease overload.

DOI： 10.1038/s41374-018-0132-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T-cell receptor (TCR) that reacts with tumor-associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR-multimers that were each directed to TAA, survivin-2B (SVN-2B) and PBF in the context of HLA-A24 (SVN-2B TCR-multimer and PBF TCR-multimer, respectively), from CTL clones that were established from peptide-vaccinated patients. Both TCR multimers could recognize cognate peptide-pulsed antigen-presenting cells, C1R-A24 cells, in a CD8-independent method. Moreover, the PBF TCR-multimer successfully recognized a PBF peptide naturally presented on HLA-A24+ PBF+ osteosarcoma cells. Taken together, the results indicated that a TCR-multimer might be useful for detection of a TAA-derived peptide presented by HLA in patients receiving immunotherapy.

DOI： 10.1111/cas.13854

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

DOI： 10.1371/journal.pone.0216503

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND AND PURPOSE: To investigate influences of proteins involved with tumor immunity on outcomes of radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC). MATERIAL AND METHODS: We performed immunohistochemical staining to examine expressions of p16 and proteins involved with tumor immunity in 92 OPSCC patients treated with radiotherapy. RESULTS: Patients with abundant infiltrating CD8-positive cells had the significantly better overall survival (OS) rate than patients with fewer CD8-positive cells (p = 0.026). Patients with higher PD-L1 expression in tumor cells (TC 1-3) had a better outcome than those with low PD-L1 expression in tumor cells (TC 0) for both OS (p = 0.019) and progression-free survival (PFS) rate (p = 0.032). Patients with high PD-L1 expression in infiltrating immune cells (IC 3) showed significantly better OS (p = 0.009) and PFS (p = 0.011) than those with low PD-L1 expression (IC 0-2). Patients with p16-negative and IC 3 showed similar OS to patients with p16-positive and IC 0-2. P16-positive tumors had a significantly higher CD8-positive cell infiltration and PD-L1 expression in tumor cells than p16-negative tumors. CONCLUSIONS: In addition to tumor p16 expression, PD-L1 expression in TC and IC can be useful for predicting the response of OPSCC to radiotherapy.

DOI： 10.1016/j.radonc.2018.08.023

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A 62-year-old woman who underwent surgery to treat pancreatic cancer provided written, informed consent to undergo adjuvant therapy with gemcitabine, tegafur, and uracil. However, this was stopped after only 14 days due to Grade 4 neutropenia. She was then started on vaccine therapy with Survivin 2B peptide (SVN-2B) including IFA and INF-α. Although metastatic lung tumors were identified and resected at 82 months after surgery, the patient has remained free of new or relapsed disease for 12 years thereafter. Tetramer and ELISPOT assays revealed the continuous circulation of SVN-2B-restricted cytotoxic T-lymphocytes (CTLs) in her peripheral blood, and CTL clones had specific activity for SVN-2B at 12 years after surgery. The adverse effects of the peptide vaccination were tolerable and comprised low-grade headache, nausea, and fatigue. A prognosis beyond 10 years in the face of pancreatic cancer with distant metastasis is extremely rare. This experience might indicate the value of cancer vaccination therapy.

DOI： 10.1007/s00262-018-2217-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: We report that the reactivity of a novel monoclonal antibody LpMab-23 for human cancer-type podoplanin (PDPN) is a predictor for a poor prognosis of tongue cancer. PATIENTS AND METHODS: The association between LpMab-23-recognizing cancer-type PDPN expression and clinical/pathological features were analyzed on 60 patients with stage I and II tongue cancer treated with transoral resection of the primary tumor. RESULTS: In the mode of invasion, the LpMab-23-dull/negative cases were significantly larger in cases with low-grade malignancies and without late cervical lymph node metastasis, than in cases with high-grade malignancies and the metastasis. In the high-grade malignant cases, LpMab-23-positive cases were significantly larger than LpMab-23-dull/negative cases. The Kaplan-Meier curves of the five-year metastasis-free survival rate (MFS) were significantly lower in the LpMab-23 positive patients than in LpMab-23 dull/negative patients. The LpMab-23-dull/negative cases showed the highest MFS in all of the clinical/pathological features and particularly, the MFS of the LpMab-23 positive cases decreased to less than 60% in the first year. In the Cox proportional hazard regression models a comparison of the numbers of LpMab-23 dull/negative with positive cases showed the highest hazard ratio with statistical significance in all of the clinical/pathological features. CONCLUSIONS: LpMab-23 positive cases may be considered to present a useful predictor of poor prognosis for early stage tongue cancer.

DOI： 10.18632/oncotarget.24986

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

DOI： 10.1111/cas.13501

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo- and radiotherapies and perpetuate tumor formation in vivo In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses. Cancer Immunol Res; 6(3); 358-69. ©2018 AACR.

DOI： 10.1158/2326-6066.CIR-17-0518

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We present a case of ovarian clear-cell carcinoma that was initially diagnosed as adenocarcinoma of lung origin. This is an instructive diagnostic pitfall for clinicians and pathologists because of the unusual clinical course, small biopsy material, and noteworthy immunophenotype of the carcinoma. Imaging analysis identified only lung and liver lesions. In addition, the biopsy specimen from the lung was TTF-1 negative and napsin A positive, which is still possible for cancer of lung origin. Postmortem examination found that the cancer should be classified as ovarian clear-cell carcinoma distinguished by positive staining for napsin A and paired-box gene 8 (PAX8). Although PAX8 may not be usually investigated when tumoral lesions are identified in only the lung and liver, it is important to keep the necessity of PAX8 in mind to excluding carcinoma of Müllerian, renal, or thyroid origin.

DOI： 10.1016/j.rmcr.2018.10.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Colorectal cancer (CRC) is a mortal disease due to treatment resistance, recurrence and distant metastasis. Emerging evidence has revealed that a small sub-population of cancer cells termed cancer stem cells (CSCs)/ cancer-initiating cells (CICs) is endowed with high levels of tumor-initiating ability, self-renewal ability and differentiation ability and is responsible for treatment resistance, recurrence and distant metastasis. Eradication of CSCs/CICs is essential to improve current treatments. However, the molecular mechanisms by which CSCs/CICs are maintained are still elusive. In this study, we aimed to determine the molecular mechanisms by which colorectal (CR)-CSCs/CICs in are maintained human primary CRC cells. CR-CSCs/CICs were isolated by sphere-culture and the ALDEFLUOR assay, and transcriptome analysis revealed that the gene ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) was expressed at high levels in CR-CSCs/CICs. Overexpression of ST6GALNAC1 enhanced the expression of sialyl-Tn (STn) antigen, which is carried by the CSC marker CD44, and increased the sphere-forming ability and resistance to a chemotherapeutic reagent. The opposite phenomena were observed by gene knockdown using siRNA. Furthermore, the Akt pathway was activated in ST6GANAC1-overexpressed cells, and activation of the pathway was cancelled by gene knockdown of galectin-3. The results indicate that ST6GALNAC1 has a role in the maintenance of CR-CSCs/CICs by activating the Akt pathway in cooperation with galectin-3 and that ST6GalNAC1 (or STn antigen) might be a reasonable molecule for CSC/CIC-targeting therapy.

DOI： 10.18632/oncotarget.22545

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVES: To identify antigenic peptides of cancer stem-like cells (CSCs) antigen, DNAJB8, and establish a mouse CSCs-targeting immunotherapy model. MATERIALS AND METHODS: To induce DNAJB8-specific immune reaction, we stimulated human CD8+ lymphocytes with antigen-presenting cells pulsed with a cocktail of three candidate HLA-A*24:02 restricted peptides and assessed peptide specific human cytotoxic T lymphocytes (CTLs) induction. One of the antigenic peptides showed identical amino acid sequence as corresponding mouse DNAJB8. We evaluated CTL induction with the peptide immunization in mouse model. RESULTS: We confirmed peptide-specific interferon-γ secretions and cytotoxic activities of induced human CTLs. In vivo immunization with the peptide to mice, peptide-specific CTL response could be observed in mouse CD8+ T cells. Furthermore, immunization with the peptide showed significant anti-tumor effects compared with negative controls. CONCLUSION: DNAJB8-derived peptide is a novel candidate for CSCs-targeting immunotherapy, and mouse models can be used to evaluate CSCs-targeting immunotherapy.

DOI： 10.1016/j.bbrc.2017.10.146

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A cell-cell adhesion protein, junctional adhesion molecule-A (JAM-A), has been shown to be involved in neoplasia of various organs. However, the fundamental role of JAM-A in tumorigenesis is still under debate because dysregulated expression of this protein has distinct effects, playing opposite roles in carcinogenesis depending on the target tissues. In the present study, we found elevated levels of JAM-A expression in lung adenocarcinoma and its preinvasive lesions, including atypical adenomatous hyperplasia and adenocarcinoma in situ by immunohistochemistry. We also showed that suppression of constitutive JAM-A expression conferred target cells with increased susceptibility to apoptosis in lung adenocarcinoma cells. Consequently, inhibition of JAM-A activity decreased colony-forming capability in vitro and tumorigenicity in vivo. The transformed phenotype following suppression of JAM-A expression was sufficient to reduce motile and invasive capacities. Importantly, knockout of JAM-A had striking effects on cells. Our observations suggest that increased expression of JAM-A promotes neoplasia of lung adenocarcinoma. In addition, an anti-JAM-A antibody efficiently reduced cell proliferation and provoked apoptosis, indicating the potential feasibility of JAM-A-inhibitory cancer therapy.

DOI： 10.1111/cas.13385

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Single cell transcriptome analysis of a cancer tissue can provide objective assessment of subtype population or the activation of each of various microenvironment component cells. In this study, we applied our newly developed technique of single cell analysis to the myometrial infiltration side (M-side) and the endometrial side (E-side) of a human endometrioid adenocarcinoma with squamous differentiation tissues. We also analyzed spherogenic cultures derived from the same tissue to identify putative regulators of stemness in vivo. Cancer cells in the E-side were highly malignant compared with those in the M-side. Many cells on the E-side were positive for spheroid-specific tumorigenesis-related markers including SOX2. In addition, there were higher numbers of epithelial-to-mesenchymal transition (EMT) cells in the E-side compared with the M-side. This study identified a site containing cells with high malignant potential such as EMT and cancer stem-like cells in cancer tissues. Finally, we demonstrate that established endometrioid adenocarcinoma subtype classifiers were variably expressed across individual cells within a tumor. Thus, such intratumoral heterogeneity may be related to prognostic implications.

DOI： 10.1038/s41598-017-14676-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Our recent work revealed that elevated expression of claudin-18 is involved in bile duct neoplasia. In the present study, we found that wound generation of a cell sheet de novo induced claudin-18 expression in its leading edge, coincident with high mitotic activity. We also found that the suppression of claudin-18 expression significantly reduced cell growth and invasiveness of bile duct cancer cell lines and tumorigenicity in vivo. In addition, an antibody specific to an extracellular loop of claudin-18 showed similar effects on the cells such as cell proliferation. Interestingly, treatment with epidermal growth factor (EGF) and overexpression of RAS oncogene induced claudin-18 expression by activation of extracellular signal-related kinase (ERK)1/2. Furthermore, enhanced claudin-18 expression activated ERK1/2. These findings provide evidence for an oncogenic property of claudin-18 in bile duct carcinoma cells via modulation of EGFR/ERK signaling, indicating that claudin-18 is a possible therapeutic target for this malignancy.

DOI： 10.1016/j.canlet.2017.05.033

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: It is essential that cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens (TAAs) and exhibit antitumor effects in immunological responses for tumor rejection. However, only a few cases with oral squamous cell carcinoma (OSCC) have been studied for these antitumor mechanisms. We established a cancer cell line and autologous CTL pair for identifying TAAs in OSCC, despite difficulties in establishing such a pair. MATERIALS AND METHODS: A cell line (OTM) from a primary lesion and a CTL line (TcOTM) were derived from OSCC in a 64-year-old female patient. CTL clones were generated by repetitive limiting dilutions. Accurate characterization was performed by in vitro analysis. RESULTS: The TcOTM clone showed specific cytotoxic activity against OTM cells in an human leukocyte antigen (HLA)-A24-restricted manner. Furthermore, it exerted cytotoxicity against the allogenic HLA-A24 cell lines. CONCLUSION: These data indicated that the TcOTM clone recognized a TAA presented by HLA-A24 cells. This unique method will allow for identification of unknown TAAs for OSCC in the future.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chemotherapy has improved the prognosis of patients with sarcomas. However, it may suppress anti-tumor immunity. Recently, we reported a novel CD8+ memory T cell population with a chemo-resistance property, "young memory" T (TYM ) cells. In this study, we investigated the proportion and function of TYM cells in peripheral blood of healthy donors and sarcoma patients who received chemotherapy and those who did not. The proportion of TYM cells was significantly decreased in patients compared with that in healthy donors. In healthy donors, anti-EBV CTLs were induced using mixed lymphocyte peptide culture, from not only TYM cells but also TCM and TEM cells. No CTLs directed to tumor-associated antigens were induced. In sarcoma patients who did not receive chemotherapy, in addition to anti-EBV CTLs, CTLs directed to the tumor-associated antigen PBF were induced from TYM , TCM and TEM cells. In sarcoma patients who received chemotherapy, EBV-specific CTLs were induced from TYM cells but were hardly induced from TEM cells. Interestingly, CTLs directed to the anti-tumor-associated antigen PBF were induced from TYM cells but not from the TCM and TEM cells in sarcoma patients who received chemotherapy. The findings suggest that TYM cells are resistant to chemotherapy and can firstly recover from the nadir. TYM cells might be important for immunological memory, especially in sarcoma patients receiving chemotherapy.

DOI： 10.1111/cas.13319

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) are defined by their higher tumor-initiating ability, self-renewal capacity and differentiation capacity. CSCs/CICs are resistant to several therapies including chemotherapy and radiotherapy. CSCs/CICs thus are thought to be responsible for recurrence and distant metastasis, and elucidation of the molecular mechanisms of CSCs/CICs are essential to design CSC/CIC-targeting therapy. In this study, we analyzed the molecular aspects of gynecological CSCs/CICs. Gynecological CSCs/CICs were isolated as ALDH1high cell by Aldefluor assay. The gene expression profile of CSCs/CICs revealed that several genes related to stress responses are preferentially expressed in gynecological CSCs/CICs. Among the stress response genes, a small heat shock protein HSP27 has a role in the maintenance of gynecological CSCs/CICs. The upstream transcription factor of HSP27, heat shock factior-1 (HSF1) was activated by phosphorylation at serine 326 residue (pSer326) in CSCs/CICs, and phosphorylation at serine 326 residue is essential for induction of HSP27. Immunohistochemical staining using clinical ovarian cancer samples revealed that higher expressions of HSF1 pSer326 was related to poorer prognosis. These findings indicate that activation of HSF1 at Ser326 residue and transcription of HSP27 is related to the maintenance of gynecological CSCs/CICs.

DOI： 10.18632/oncotarget.16361

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.

DOI： 10.1016/j.bbrc.2017.02.112

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are small sub-population of cancer cells that are endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs could be isolated as high aldehyde dehydrogenase 1 activity cells (ALDH1high) from various cancer samples. In this study, we isolated urothelial carcinoma CSCs/CICs as ALDHhigh cells and investigated the molecular aspects. ALDH1high cells showed greater sphere-forming ability and higher tumor-initiating ability in immune-deficient mice than those of ALDH1low cells, indicating that CSCs/CICs were enriched in ALDH1high cells. cDNA microarray analysis revealed that an ionotropic glutamate receptor glutamate receptor, ionotropic, kainate 2 (GRIK2) was expressed in ALDH1high cells at a higher level than that in ALDH1low cells. GRIK2 gene knockdown by siRNAs decreased the sphere-forming ability and invasion ability, whereas GRIK2 overexpression increased the sphere-forming ability, invasion ability and tumorigenicity, indicating that GRIK2 has a role in the maintenance of CSCs/CICs. Immunohistochemical staining revealed that higher levels of GRIK2 and ALDH1 expression were related to poorer prognosis in urinary tract carcinoma cases. The findings indicate that GRIK2 has a role in the maintenance of urothelial CSCs/CICs and that GRIK2 and ALDH1 can be prognosis prediction markers for urinary tract carcinomas.

DOI： 10.18632/oncotarget.16259

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Many human cancers have been reported to have enhanced expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1), which binds to programmed cell death-1 (PD-1) expressed on immune cells. PD-L1/PD-1 plays a role in inhibition of antitumor immunity by inducing T cell apoptosis and tolerance. Thus, it is crucial to elucidate mechanisms of PD-L1 expression on cancer cells. ERO1-α is an oxidase located in the endoplasmic reticulum. It is overexpressed in a variety of tumor types and it plays a role in disulfide bond formation in collaboration with PDI. Here, we investigated the influence of ERO1-α on expression of PD-L1 and immune escape. We demonstrated that ERO1-α augmented the expression of PD-L1 via facilitation of oxidative protein folding within PD-L1. In addition, we showed that overexpression of ERO1-α increased HIF-1α protein expression, resulting in an increase of PD-L1 mRNA as well as protein. In clinical cases, we observed that the expression of ERO1-α in triple negative breast cancer was related to the expression of PD-L1. Moreover, apoptosis of Jurkat leukemia T cells, which express PD-1, induced by tumor PD-L1 was inhibited when ERO1-α was depleted. The results suggest that targeting ERO1-α in tumor cells can be a novel approach for cancer immunotherapy. Therefore, the role of ERO1-α in tumor-mediated immunosuppression should be further explored.

DOI： 10.18632/oncotarget.14960

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Colorectal cancer (CRC) is one of the most common malignancy, and the prognosis is not still satisfactory due to treatment resistance, recurrence and distant metastasis. Cancer stem cells (CSCs)/cancer-initiating cells (CICs) is endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability, and CSCs/CICs are resistant to treatments. Thus, CSCs/CICs are thought to be responsible for recurrence and distant metastasis, and eradication of CSCs/CICs is essential to cure CRCs. However, the molecular mechanisms of CSCs/CICs are remain unknown, and we aimed to elucidate molecular aspects of CR-CSCs/CICs in this study. We screened the transcriptome data of primary human CR-CSCs/CICs that we previously established, and found that LEM domain containing 1 (LEMD1) is preferentially expressed in CR-CSCs/CICs. LEMD1 belongs to cancer-testis (CT) antigen, and has five transcript variants (variant 1 [V1] - variant 5 [V5]). We found that LEMD1 V1, V2 and V3 is expressed in testis and CR-CSCs/CICs, whereas LEMD1 V4 and V5 is ubiquitously expressed. LEMD1 gene knockdown experiments using siRNAs and gene overexpression experiments revealed that LEMD1 has a role in the maintenance of CR-CSCs/CICs. These observations indicate that CR-CSC/CIC-specific LEMD1 variants are reasonable target of CR-CSC/CIC-targeted therapy.

DOI： 10.1016/j.bbrc.2017.02.081

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Accumulating evidence indicates that immune checkpoint inhibition-mediated cancer immunotherapies greatly improve the prognosis of certain types of cancer. This approach is now becoming a standard therapy, joining surgery, radiotherapy, and chemotherapy. Because the costs of antibody drugs are now a socioeconomic burden in many countries, an urgent need in cancer immunotherapy is the identification of relevant biomarkers that can predict therapy efficacy. Recent studies have reported that colorectal adenocarcinoma with hereditary or sporadic deficiency in mismatch repair (MMR) proteins has high antigenicity and that detection of these proteins could be a promising way to estimate clinical response. In this study of 135 patients with colorectal cancer, we used immunohistochemistry to investigate the correlation between deficiency in MMR proteins and expression of human leukocyte antigen (HLA) class I molecules, a prerequisite of cytotoxic T-cell-based immunotherapy. Interestingly, MMR protein deficiency was an independent risk factor for the impaired expression of HLA class I molecules (odds ratio (OR)=10.44, 95% confidence interval (CI)=3.15-34.62, p<0.001), suggesting the existence of a putative entity that we have named "adaptive immune escape". Moreover, our results might provide a potential novel biomarker for the selection of patients who would respond to cancer immunotherapies. At the same time, the results suggest that we have to overcome the impaired expression of HLA class I molecules to further improve the cure rate of cancer immunotherapies.

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Identification of tumor-associated antigens (TAAs) recognized by T cells has enabled clinical peptide vaccination trials targeting TAAs to be performed for patients with carcinoma or sarcoma. Although peptide vaccination could elicit specific immunological responses, clinical objective responses were not frequently observed, especially in end-stage patients with large tumor burdens who were receiving high-dose chemotherapy. The key points for developing effective peptide vaccination therapy are (i) targeting cancer stem-like cell antigens that are expressed in cancer cells but not in normal cells and regulating tumorigenesis and (ii) manip- ulating memory T stem cells that have the capacity of long-living and resistance to chemo- therapeutic drugs. The combination of a peptide vaccination and immune checkpoint inhibi- tors might also be attractive.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells.

DOI： 10.1371/journal.pone.0171460

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC-MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8+ T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.

DOI： 10.1080/2162402X.2017.1293214

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cytotoxic T-lymphocytes (CTLs) lyse target cells after recognizing the complexes of peptides and MHC class I molecules (pMHC I) on cell surfaces. Tapasin is an essential component of the peptide-loading complex (PLC) and its absence influences the surface repertoire of MHC class I peptides. In the present study, we assessed tapasin expression in 85 primary tumor lesions of non-small cell lung cancer (NSCLC) patients, demonstrating that tapasin expression positively correlated with patient survival. CD8+ T-cell infiltration of tumor lesions was synergistically observed with tapasin expression and correlated positively with survival. To establish a direct link between loss of tapasin and CTL recognition in human cancer models, we targeted the tapasin gene by CRISPR/Cas9 system and generated tapasin-deficient variants of human lung as well as colon cancer cells. We induced the CTLs recognizing endogenous tumor-associated antigens (TAA), survivin or cep55, and they responded to each tapasin-proficient wild type. In contrast, both CTL lines ignored the tapasin-deficient variants despite their antigen expression. Moreover, the adoptive transfer of the cep55-specific CTL line failed to prevent tumor growth in mice bearing the tapasin-deficient variant. Loss of tapasin most likely limited antigen processing of TAAs and led to escape from TAA-specific CTL recognition. Tapasin expression is thus a key for CTL surveillance against human cancers.

DOI： 10.1080/2162402X.2016.1274476

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chest wall tumors are relatively rare, and hematogenous metastasis to the chest wall is very rare. We herein describe a rare case of occult thyroid carcinoma as metastasis to the chest wall in an 80-year-old woman. The patient received detailed examinations of the chest wall tumor, and the results suggested that she had occult thyroid carcinoma. Surgery was then performed to remove all of her thyroid. As a result, she was diagnosed with follicular carcinoma of the thyroid. We report an extremely rare case of occult thyroid carcinoma diagnosed as hematogenous metastasis to the chest wall.

DOI： 10.2169/internalmedicine.56.8340

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Cancer-stem cells (or cancer-stem like cells, CSC) play an indispensable role in tumor initiation or tumor development in vivo. However, CSCs are resistant to conventional therapies including chemo/radiotherapy and a certain molecularly-targeted therapy, thereby are responsible for tumor relapse or metastasis in clinical settings. In this review, we focus on cytotoxic T-cell mediated immune responses against CSCs and discuss a challenge of targeting CSCs as well as the development of CSC-based cancer immunotherapy, which is an emerging and promising strategy toward a complete cure of quite a few types of cancers.

DOI： 10.2177/jsci.40.40

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-α plays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-α facilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

DOI： 10.1002/eji.201646525

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The aim of peptide vaccination therapy is to stimulate and activate peptide-specific T cells to reject tumor cells. This strategy has been promoted by the discovery of tumor-associated antigens recognized by T cells. Peptide vaccination therapy can induce immune responses in some cancer patients but the objective clinical response rates are still low. To improve of the efficacy of peptide vaccination therapy: (i) cancer stem cell antigens specifically expressed in carcinoma/sarcoma stem-like cells but not in normal cells are needed; (ii) peptide vaccination therapy should be performed in the earlier stages; and (iii) memory T stem cells should be regulated to maintain long-lasting immune responses to the peptide vaccination.

DOI： 10.1111/pin.12438

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.

DOI： 10.18632/oncotarget.10571

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

INTRODUCTION: The use of immunotherapeutic challenges for sarcoma has a long history. Despite the existence of objective responses, immunotherapy has been overshadowed by the results of chemotherapy, especially for osteosarcoma. However, the prognosis for non-responders to chemotherapy is still poor and immunotherapy is now focused on again. AREAS COVERED: We reviewed the following types of clinical trials of immunotherapy for sarcoma: (i) vaccination with autologous tumor cells, (ii) vaccination with peptides derived from tumor-associated antigens, (iii) adoptive cell transfer using engineered T cells expressing T cell receptor directed at NY-ESO-1 and (iv) immune checkpoint inhibitors targeting CTLA-4 and PD1/PDL1. EXPERT OPINION: The immunogenicity of sarcoma might be lower than that of melanoma. Patients with small lesions who have not received any chemotherapy are good candidates for peptide-based immunotherapy. Combining peptide vaccination and immune checkpoint inhibitors is an attractive option, and long-lived memory T cells are attracting attention. Memory T stem cells defined by CD95+ are long-lived and have the capacity for self-renewal and multidifferentiation. We also identified a novel memory T cell population, young memory T cells defined by CD73+CXCR3+. Regulation of such memory T stem cells will be useful for peptide vaccination and adoptive cell transfer.

DOI： 10.1080/14712598.2016.1188075

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記述言語：英語  

DOI： 10.1016/j.ebiom.2016.07.009

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy. EXPERIMENTAL DESIGN: Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model. RESULTS: OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model. CONCLUSIONS: OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; 22(13); 3298-309. ©2016 AACR.

DOI： 10.1158/1078-0432.CCR-15-1709

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

DOI： 10.1016/j.bbrc.2016.03.152

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

AIM: Sex-determining region Y-box binding protein-2 (SOX2) and aldehyde dehydrogenase-1 (ALDH1) are known cancer stem-cell markers, and represent candidate predictors for breast cancer prognosis. In this study we investigated the relationships between SOX2/ALDH1 expression and prognosis. MATERIALS AND METHODS: One hunred and two breast cancer surgical specimens were immunohistochemically analyzed for SOX2 and ALDH1 expression. RESULTS: Disease-free survival (DFS) and overall survival (OS) were significantly poorer for SOX2-positive patients than SOX2-negative (p=0.0024 and p=0.0021, respectively), and for ALDH1-positive patients than ALDH1-negative (p=0.0049 and p=0.0083). DFS and OS were worse for SOX2- or ALDH1-positive patients than double-negative (p=0.0053 and p=0.0166). While an obvious tendency toward worse DFS was seen for estrogen receptor (ER)-negative patients, and attenuated for ER-positive, only SOX2/ALDH1 any-positive patients showed significantly poorer DFS (p=0.0258). CONCLUSION: SOX2 and ALDH1 can be considered markers of poor prognosis, particularly in ER-negative patients. SOX2/ALDH1 any-positivity might also offer a reliable predictor of poor prognosis.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

High-dose chemotherapy may kill not only tumor cells but also immunocytes, and frequently induces severe lymphocytopenia. On the other hand, patients who recover from the nadir maintain immunity against infection, suggesting the existence of an unknown memory T-cell population with stress resistance, long-living capacity, proliferation and differentiation. Recently, the differentiation system of T-cell memory has been clarified using mouse models. However, the human T-cell memory system has great diversity induced by natural antigens derived from many pathogens and tumor cells throughout life, and profoundly differs from the mouse memory system constructed using artificial antigens and transgenic T cells. Here we report a novel human T-cell memory population, "young memory" T (TYM) cells. TYM cells are defined by positive expression of CD73, which represents high aldehyde dehydrogenase 1 (ALDH1) activity and CXCR3 among CD8(+)CD45RA(+)CD62L(+) T cells. TYM proliferate upon TCR stimulation, with differentiation capacity into TCM and TEM and drug resistance. Moreover, TYM are involved in memory function for viral and tumor-associated antigens in healthy donors and cancer patients, respectively. Regulation of TYM might be very attractive for peptide vaccination, adoptive cell-transfer therapy and hematopoietic stem cell transplantation.

DOI： 10.1080/2162402X.2016.1165376

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Endoplasmic reticulum disulfide oxidase 1-α (ERO1-α) is an oxidase that exists in the endoplasmic reticulum and has a role in the formation of disulfide bonds of secreted proteins and cell-surface proteins. Recently, we reported that ERO1-α is present in high levels in various types of tumours, and that ERO1-α is a novel factor of poor prognosis. However, how ERO1-α affects a tumour in vivo and why patients who have a tumour with a high expression level of ERO1-α have a poor prognosis are still unknown. Therefore, to clarify the mechanism, we investigated the effect of ERO1-α on a tumour from the point of view of angiogenesis. METHODS: The effect of ERO1-α on tumour growth and angiogenesis was analysed by using non-obese diabetic-severe combined immunodeficient mice. The production of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells with ERO1-α- overexpression or with ERO1-α knockdown was measured. The role of ERO1-α on VEGF expression was investigated. In triple-negative breast cancer cases, the relationship between expression of ERO1-α and angiogenesis was analysed. RESULTS: We found that the expression of ERO1-α promoted tumour growth in a mouse study and angiogenesis. The effects of ERO1-α on angiogenesis were mediated via oxidative protein folding of VEGF and enhancement of VEGF mRNA expression by using MDA-MB-231. In triple-negative breast cancer cases, the expression of ERO1-α related to the number of the blood vessel. Furthermore, we found that ERO1-α was a poor prognosis factor in triple-negative breast cancer. CONCLUSIONS: Our study has established a novel link between expression of ERO1-α and secretion of VEGF, providing new evidence for the effectiveness of ERO1-α-targeted therapy in patients with ERO1-α-expressed cancer.

DOI： 10.1038/bjc.2016.105

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Skin atrophy and delayed wound healing are observed in aged humans; however, the molecular mechanism are still elusive. The aim of this study was to analyze the molecular mechanisms of delayed wound healing by aging using α-Klotho-deficient (kl/kl) mice, which have phenotypes similar to those of aged humans. The kl/kl mice showed delayed wound healing and impaired granulation formation compared with those in wild-type (WT) mice. The skin graft experiments revealed that delayed wound healing depends on humoral factors, but not on kl/kl skin tissue. The mRNA expression levels of cytokines related to acute inflammation including IL-1β, IL-6 and TNF-α were higher in wound lesions of kl/kl mice compared with the levels in WT mice by RT-PCR analysis. LPS-induced TNF-α production model using spleen cells revealed that TNF-α production was significantly increased in the presence of FGF23. Thus, higher levels of FGF23 in kl/kl mouse may have a role to increase TNF-α production in would lesion independently of α-Klotho protein, and impair granulation formation and delay wound healing.

DOI： 10.1016/j.bbrc.2016.03.138

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Epithelial ovarian cancer (EOC) is one of the most lethal cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) have been reported to be origin of primary and recurrent cancers and to be resistant to several treatments. In this study, we identified matrix metalloproteinase-10 (MMP10) is expressed in CSCs/CICs of EOC. An immunohistochemical study revealed that a high expression level of MMP10 is a marker for poor prognosis and platinum resistance in multivariate analysis. MMP10 gene overexpression experiments and MMP10 gene knockdown experiments using siRNAs revealed that MMP10 has a role in the maintenance of CSCs/CICs in EOC and resistance to platinum reagent. Furthermore, MMP10 activate canonical Wnt signaling by inhibiting noncanonical Wnt signaling ligand Wnt5a. Therefore, MMP10 is a novel marker for CSCs/CICs in EOC and that targeting MMP10 is a novel promising approach for chemotherapy-resistant CSCs/CICs in EOC.

DOI： 10.18632/oncotarget.8645

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as small subpopulation of cancer cells that are endowed with higher tumor-initiating ability. CSCs/CICs are resistant to standard cancer therapies including chemotherapy and radiotherapy, and they are thus thought to be responsible for cancer recurrence and metastasis. Therefore, elucidation of molecular mechanisms of CSCs/CICs is essential to cure cancer. In this study, we analyzed the gene expression profiles of gynecological CSCs/CICs isolated as aldehyde dehydrogenase high (ALDH(high)) cells, and found that MAPK13, PTTG1IP, CAPN1 and UBQLN2 were preferentially expressed in CSCs/CICs. MAPK13 is expressed in uterine, ovary, stomach, colon, liver and kidney cancer tissues at higher levels compared with adjacent normal tissues. MAPK13 gene knockdown using siRNA reduced the ALDH(high) population and abrogated the tumor-initiating ability. These results indicate that MAPK13 is expressed in gynecological CSCs/CICs and has roles in the maintenance of CSCs/CICs and tumor-initiating ability, and MAPK13 might be a novel molecular target for treatment-resistant CSCs/CICs.

DOI： 10.1016/j.bbrc.2016.03.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition.

DOI： 10.1002/eji.201545835

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cervical cancer is a major cause of cancer death in females worldwide. Cervical cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are resistant to conventional radiotherapy and chemotherapy, and CSCs/CICs are thought to be responsible for recurrence. Eradication of CSCs/CICs is thus essential to cure cervical cancer. In this study, we isolated cervical CSCs/CICs by sphere culture, and we identified a cancer testis (CT) antigen, CTCFL/BORIS, that is expressed in cervical CSCs/CICs. BORIS has 23 mRNA isoform variants classified by 6 subfamilies (sfs), and they encode 17 different BORIS peptides. BORIS sf1 and sf4 are expressed in both CSCs/CICs and non-CSCs/CICs, whereas BORIS sf6 is expressed only in CSCs/CICs. Overexpression of BORIS sf6 in cervical cancer cells increased sphere formation and tumor-initiating ability compared with those in control cells, whereas overexpression of BORIS sf1 and BORIS sf4 resulted in only slight increases. Thus, BORIS sf6 is a cervical CSC/CIC-specific subfamily and has a role in the maintenance of cervical CSCs/CICs. BORIS sf6 contains a specific c-terminal domain (C34), and we identified a human leukocyte antigen (HLA)-A2-restricted antigenic peptide, BORIS C34_24(9) encoded by BORIS sf6. A BORIS C34_24(9)-specific cytotoxic T cell (CTL) clone showed cytotoxicity for BORIS sf6-overexpressing cervical cancer cells. Furthermore, the CTL clone significantly suppressed sphere formation of CaSki cells. Taken together, the results indicate that the CT antigen BORIS sf6 is specifically expressed in cervical CSCs/CICs, that BORIS sf6 has a role in the maintenance of CSCs/CICs, and that BORIS C34_24(9) peptide is a promising candidate for cervical CSC/CIC-targeting immunotherapy.

DOI： 10.18632/oncotarget.7165

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pancreatic tumors are chemoresistant and malignant, and there are very few therapeutic options for pancreatic cancer, as the disease is normally diagnosed at an advanced stage. Although attempts have been made to develop vaccine therapies for pancreatic cancer for a couple of decades, none of the resultant protocols or regimens have succeeded in improving the clinical outcomes of patients. We herein review vaccines tested within the past few years, including peptide, biological and multiple vaccines, and describe the three sets of criteria used to evaluate the therapeutic activity of vaccines in solid tumors.

DOI： 10.1007/s00595-015-1120-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSC)/cancer-initiating cells (CIC) are defined as minor subpopulations of cancer cells that are endowed with properties of higher tumor-initiating ability, self-renewal ability and differentiation ability. Accumulating results of recent studies have revealed that CSC/CIC are resistant to standard cancer therapies, including chemotherapy, radiotherapy and molecular targeting therapy, and eradiation of CSC/CIC is, thus, critical to cure cancer. Cancer immunotherapy is expected to become the "fourth" cancer therapy. Cytotoxic T lymphocytes (CTL) play an essential role in immune responses to cancers, and CTL can recognize CSC/CIC in an antigen-specific manner. CSC/CIC express several tumor-associated antigens (TAA), and cancer testis (CT) antigens are reasonable sources for CSC/CIC-targeting immunotherapy. In this review article, we discuss CSC/CIC recognition by CTL, regulation of immune systems by CSC/CIC, TAA expression in CSC/CIC, and the advantages of CSC/CIC-targeting immunotherapy.

DOI： 10.1111/cas.12830

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined by their abilities of tumor initiation, self-renewal and differentiation. In a previous study, we showed by gene knockdown using siRNA and gene overexpression experiments that Dnaj (Hsp40) homolog, subfamily B, member 8 (DNAJB8), a role in the maintenance, of renal cell carcinoma CSCs/CICs. In the present study, we established Dnajb8 knockout (KO) renal cell carcinoma (RCC) line cells (RenCa cells) and analyzed the cells to confirm the function of Dnajb8 in RCC CSCs/CICs. Dnajb8 KO cells showed reduced ratios of side population cells and reduced sphere forming ability. An in vivo single cell tumor initiation assay revealed that the numbers of CSCs/CICs were 3 in 4 wild-type RenCa cells and 1 in 4 Dnajb8 KO cells. Dnajb8 KO cells showed sensitivity to Docetaxel. On the other hand, Dnajb8 KO cells did not show any sensitivities to stresses including low pH, low glucose, heat shock and sensitivity to cisplatin. The results indicate that Dnajb8 has a role in tumor initiation, side population ratio and sphere formation but it is dispensable for stress responses.

DOI： 10.1371/journal.pone.0146501

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Extensive research over 100 years has clarified that cancers are surely suppressed by human immune system. Unfortunately, a number of clinical trials with various approaches have fallen short of clinical application because of the lack of significant anti-tumor effect. However, recent approved immune checkpoint inhibitors, namely PD-1 and CTLA-4 blockers, provide even better prognosis than existing chemotherapy in patients with certain types of cancer. There is no doubt that immunotherapy is becoming a standard treatment as well as surgery, chemotherapy and radiotherapy. Dendritic cells express a variety of immune checkpoint molecules including one with unknown function. In addition, accumulating evidence reported that already known molecules might have alternative functions. Further investigation of this field would lead to development of more effective treatments for cancer with less adverse effects. In this short review, we introduce some immune checkpoint molecules expressed in dendritic cells from the point of view of cancer immunotherapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

DOI： 10.1371/journal.pone.0158903

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Increasing evidence indicates that cancer stem cells have essential roles in tumor initiation, progression, metastasis and resistance to chemo-radiation. Recent research has pointed out biological importance of microRNAs in cancer stem cell dysregulation. Total number of mature microRNAs in human genome increased to more than 2,500 with the recent up-date of the database. However, currently no information is available regarding microRNA expression profiles of cancer stem cells in head and neck squamous cell carcinoma (HNSCC). Increased ALDH1 activity has been demonstrated as a reliable marker for isolation of cancer stem cells. Therefore, we evaluated the microRNA expression profile of ALDH1-high subpopulations in the HNSCC cell lines UTSCC-9 and UTSCC-90. Initially, we examined cancer stem cell properties of ALDH1-high subpopulations in both cell lines. We analyzed expression of stemness markers, sphere formation capacity and xenograft transplantation into NOD/SCID mice. Our findings validated that ALDH1-high subpopulations showed significantly increased tumor-initiating ability. Furthermore, we investigated the microRNA expression profile of HNSCC stem cells using microRNA array and confirmed the results by quantitative real-time PCR. We found that expressions of miR‑424, let-7a, miR‑6836, miR‑6873 and miR‑7152 were downregulated, whereas miR‑147b was upregulated with statistical significance in the ALDH1-high subpopulation. In conclusion, we identified a subset of microRNAs that were differentially expressed in ALDH1-high subpopulation, providing new microRNA targets to study dysregulation of HNSCC-initiating cells and develop therapeutic strategies aimed at eradicating the tumorigenic stem cells in HNSCC.

DOI： 10.3892/ijo.2015.3145

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Plasmacytoid dendritic cells (pDCs) are the main producers of IFN-α in response to unmethylated DNA molecules, including cytosine guanine dinucleotide (CpG)-DNA in vivo. pDCs specifically express toll-like receptor (TLR) 9 and are therefore able to recognize the unmethylated DNAs. It has recently been shown that not only conventional DCs (cDCs) but also pDCs efficiently cross-present exogenous antigens after TLR9 activation. However, the precise molecular mechanism has remained unclear. Here, we show that pDCs secreted heat shock protein 72 (Hsp72) in response to CpG-A administration in a TLR9-dependent manner. Extracellular Hsp72 bound to an Hsp90-peptide complex and enhanced binding of Hsp90-peptide complex to pDC, resulting in efficient cross-presentation. Our experiments therefore suggest a mechanism for orchestration of immune responses by stimulation of pDCs with CpG-A.

DOI： 10.1016/j.imlet.2015.06.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with β2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.

DOI： 10.4049/jimmunol.1303228

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cytotoxic T lymphocytes (CTLs) recognize the human leukocyte antigen (HLA) class I and antigenic peptide complex, and they play a crucial role in cancer immunity. Our recent study revealed that HLA class I downregulation is related to poorer prognosis and a low level of intratumoral CTLs is associated with platinum resistance, indicating the significance of immunological surveillance.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

CD4(+) T cell effectors are crucial for establishing antitumor immunity. Dendritic cell maturation by immune adjuvants appears to facilitate subset-specific CD4(+) T cell proliferation, but the adjuvant effect for CD4 T on induction of cytotoxic T lymphocytes (CTLs) is largely unknown. Self-antigenic determinants with low avidity are usually CD4 epitopes in mutated proteins with tumor-associated class I-antigens (TAAs). In this study, we made a chimeric version of survivin, a target of human CTLs. The chimeric survivin, where human survivin-2B containing a TAA was embedded in the mouse survivin frame (MmSVN2B), was used to immunize HLA-A-2402/K(b)-transgenic (HLA24(b)-Tg) mice. Subcutaneous administration of MmSVN2B or xenogeneic human survivin (control HsSNV2B) to HLA24(b)-Tg mice failed to induce an immune response without co-administration of an RNA adjuvant polyI:C, which was required for effector induction in vivo. Although HLA-A-2402/K(b) presented the survivin-2B peptide in C57BL/6 mice, 2B-specific tetramer assays showed that no CD8(+) T CTLs specific to survivin-2B proliferated above the detection limit in immunized mice, even with polyI:C treatment. However, the CD4(+) T cell response, as monitored by IFN-γ, was significantly increased in mice given polyI:C+MmSVN2B. The Th1 response and antibody production were enhanced in the mice with polyI:C. The CD4 epitope responsible for effector function was not Hs/MmSNV13-27, a nonconserved region between human and mouse survivin, but region 53-67, which was identical between human and mouse survivin. These results suggest that activated, self-reactive CD4(+) helper T cells proliferate in MmSVN2B+polyI:C immunization and contribute to Th1 polarization followed by antibody production, but hardly participate in CTL induction.

DOI： 10.1016/j.imbio.2014.08.017

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously found that prostate cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) express hepatocyte growth factor (HGF) and that the HGF/c-MET proto-oncogene product (c-MET) signal has a role in the maintenance of prostate CSCs/CICs in an autocrine fashion. HGF is, thus, a novel marker for prostate CSCs/CICs. We hypothesized that high expression of HGF might be related to early recurrence of prostate cancer after radical prostatectomy, and the purpose of the present study was to evaluate the relationship between expression of HGF in prostate tissues and biochemical recurrence after radical prostatectomy. One hundred-one patients with prostate cancer who underwent open or laparoscopic radical prostatectomy from November 2008 to October 2011 with an adequate prostate-specific antigen (PSA) follow-up period, were investigated. Immunohistochemical staining of HGF was compared to biochemical recurrence after radical prostatectomy. Patients with tumors exhibiting HGF positivity of 5% or more had a significantly shorter biochemical recurrence-free period than that of patients whose tumor HGF positivity was less than 5% (p=0.001). In multivariate Cox regression, preoperative PSA and HGF positivity were independent predictors of biochemical recurrence following prostatectomy. Our finding suggests a direct link between expression of HGF, a novel prostate marker of CSCs/CICs, and biochemical recurrence after radical prostatectomy in patients with prostate cancer.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Spontaneous regression (SR) is defined as the complete or partial disappearance of disease without anticancer treatments. We report a case of SR of small cell lung cancer (SCLC) combined with cancer associated retinopathy (CAR). A 65-year-old woman was admitted to our hospital to examine abnormal shadows of the lung with visual loss. She was diagnosed with SCLC associated with CAR. Subsequent chest X-ray and CT scan showed partial regression of both primary tumor and lymph node metastasis without anticancer treatment. Recoverin antigen was present on the tumor cells and anti-recoverin antibody was observed in the patient's serum. Activation of recoverin-specific antitumor cytotoxic T lymphocyte (CTL) was observed in this patient. SCLC was considered to reduce spontaneously by the activation of recoverin-specific antitumor CTL. To the best of our knowledge, this is the first report of SR in SCLC combined with CAR.

DOI： 10.1016/j.lungcan.2014.10.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The presentation of an exogenous antigen in a major histocompatibility complex class-I- restricted fashion to CD8(+) T cells is called cross-presentation. Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit efficient CTL responses by cross-presentation through an as-yet entirely unknown mechanism. Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We have shown that a tumor antigen peptide complexed with Hsp90 could be cross-presented by dendritic cells (DCs) through an endosomal pathway in a murine system. However, it has not been determined whether human DCs also cross-present an Hsp90-peptide complex and induce peptide-specific CTLs. In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway. Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

DOI： 10.1111/cas.12570

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Epithelial ovarian cancer (EOC) is one of the most deadly carcinomas in females. Immune systems can recognize EOCs; however, a defect of human leukocyte antigen (HLA) class I expression is known to be a major mechanism for escape from immune systems, resulting in poor prognosis. The purpose of this study is to identify novel correlations between immunologic responses and other clinical factors. We investigated the expression of immunologic components in 122 cases of EOCs for which surgical operations were performed between 2001 and 2011. We immunohistochemically stained EOC specimens using an anti-pan HLA class I monoclonal antibody (EMR8-5) and anti-CD3, -CD4, and -CD8 antibodies, and we analyzed correlations between immunologic parameters and clinical factors. In multivariate analysis that used the Cox proportional hazards model, independent prognostic factors for overall survival in advanced EOCs included low expression level of HLA class I [risk ratio (RR), 1.97; 95% confidence interval (CI), 1.01-3.83; P = 0.046] and loss of intraepithelial cytotoxic T lymphocyte (CTL) infiltration (RR, 2.11; 95% CI, 1.06-4.20; P = 0.033). Interestingly, almost all platinum-resistant cases showed a significantly low rate of intraepithelial CTL infiltration in the χ(2) test (positive vs. negative: 9.0% vs. 97.7%; P < 0.001). Results from a logistic regression model revealed that low CTL infiltration rate was an independent factor of platinum resistance in multivariate analysis (OR, 3.77; 95% CI, 1.08-13.12; P = 0.037). Platinum-resistant EOCs show poor immunologic responses. The immune escape system of EOCs may be one of the mechanisms of platinum resistance.

DOI： 10.1158/2326-6066.CIR-14-0101

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cells within cancer that contribute to cancer initiation and progression. Cancer-associated fibroblasts (CAFs) are stromal fibroblasts surrounding tumor cells, and they have important roles in tumor growth and tumor progression. It has been suggested that stromal fibroblasts and CSCs/CICs might mutually cooperate to enhance their growth and tumorigenic capacity. In this study, we investigated the effects of fibroblasts on tumor-initiating capacity and stem-like properties of ovarian CSCs/CICs. CSCs/CICs were isolated from the ovarian carcinoma cell line HTBoA as aldehyde dehydrogenase 1 high (ALDH1(high)) population by the ALDEFLUOR assay. Histological examination of tumor tissues derived from ALDH1(high) cells revealed few fibrous stroma, whereas those derived from fibroblast-mixed ALDH1(high) cells showed abundant fibrous stroma formation. In vivo tumor-initiating capacity and in vitro sphere-forming capacity of ALDH1(high) cells were enhanced in the presence of fibroblasts. Gene expression analysis revealed that fibroblast-mixed ALDH1(high) cells had enhanced expression of fibroblast growth factor 4 (FGF4) as well as stemness-associated genes such as SOX2 and POU5F1. Sphere-forming capacity of ALDH1(high) cells was suppressed by small-interfering RNA (siRNA)-mediated knockdown of FGFR2, the receptor for FGF4 which was expressed preferentially in ALDH1(high) cells. Taken together, the results indicate that interaction of fibroblasts with ovarian CSCs/CICs enhanced tumor-initiating capacity and stem-like properties through autocrine and paracrine FGF4-FGFR2 signaling.

DOI： 10.1038/labinvest.2014.122

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy.

DOI： 10.1111/cas.12362

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: We previously found that hypoxia-inducible factor (HIF) prolyl hydroxylase-3 (PHD3) was frequently overexpressed in renal cell carcinomas (RCCs), unlike in normal tissues, and therefore, we studied the mechanism and role of PHD3 expression in RCC. METHODS: The von Hippel-Lindau (VHL)-gene-mutant RCC cell lines SMKT-R2 and SMKT-R3 and wild-type VHL cell lines Caki-1 and ACHN were used. Associations of the expression of PHD3 with HIF-α proteins and signal transduction pathways were evaluated under normoxic conditions. The effect of PHD3 on cell proliferation was also examined by small interference RNA and cDNA transfection. Moreover, the prognostic impact of PHD3 expression in clear cell RCC (CCRCC) was evaluated using primary cancer tissues. RESULTS: In SMKT-R2 and SMKT-R3, HIF-α proteins were expressed and PHD3 was highly expressed. On the other hand, ACHN had low expression of HIF-α proteins and PHD3. However, Caki-1 had high expression of PHD3 even though there was no distinct expression of HIF-α proteins. PHD3 expression was inhibited by blockade of Akt and mammalian target of rapamycin (mTOR), but not by HIF-1α and HIF-2α double knockdown. In addition, PHD3 knockdown resulted in the promotion of cell proliferation in SMKT-R2, SMKT-R3 and Caki-1. On the other hand, forced expression of PHD3 reduced cell proliferation in ACHN. In immunohistochemistry, PHD3 expression was a significant factor for better recurrence-free survival in patients with CCRCC. CONCLUSIONS: PHD3 expression can be induced by the phosphatidylinositol-3 kinase/Akt/mTOR pathway in RCC independently of HIF proteins. Furthermore, PHD3 has an antiproliferative function independent of HIF protein status in RCC, indicating a novel expression mechanism and function of PHD3.

DOI： 10.1007/s00432-014-1593-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Identification of antigenic peptides derived from tumor-associated antigens (TAA) enables cancer vaccine therapy using antigenic peptides. Here, we summarize the design of antigenic peptides and induction of cytotoxic T lymphocytes (CTL) using antigenic peptides and validation of CTL.

DOI： 10.1007/978-1-4939-0345-0_28

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Tumor immunology has been advancing after the great discovery of tumor-specific antigen MAGE in 1991, and a number of tumor antigens have been reported to date. We have also found novel tumor antigens through various methodologies such as gene expression cloning, bioinformatics, reverse immunology, transcriptome analysis and peptidome analysis. Recently, we made a success of defining cancer stem cell-specific antigens. The fruits of our basic research have been applied to clinical trials of cancer vaccine. The long path and future perspectives of innovative immunotherapeutic drug development are described.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Circulating CD8+ T cells (CTL) survey the peptide/MHC class I complexes on cell surface to discriminate self and eliminate foreign/transformed cells. Thus, the MHC class I peptide repertoire directly influences cells fate, and provides information for immunotherapy strategy. Here we target HLA-A24 (A24), the most frequent serotype in Asia, and perform large-scale mass spectrometric profiling of natural peptides. Using the antibody specific to A24, we identified 264 peptides from colon (SW480, Colo320, HCT15/b2m) and 331 peptides from lung (LHK2, Sq-1) cancer cells. Although, known A24 binding anchors (Y/F at P2 and F/L/I at P9) are strongly conserved among the detected ligands, a subset of peptides with an unusual anchor (K/R at the C-terminal P9 or P10) is observed, suggesting diverse usage of anchors in certain types of cancer cells. Moreover, some peptides (and their genes) are exclusively expressed in cancer stem cells (cells able to exclude Hoechst dye). In summary, we identified approx. 500 non-overlapping natural peptides presented by A24 from colon and lung cancer cells. A combination of natural peptides specific to tumors could be an ideal way for a CTL-based immunotherapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein overexpressed only in the prostate among normal tissues and various types of cancer including prostate, bladder, lung, and ovarian cancer. Although its function in prostate and tumor cells has been remained unclear, due to its unique and restricted expression, STEAP-1 is expected to be an attractive target for cancer therapy. Here, we show that knockdown of STEAP-1 in human cancer cells caused the retardation of tumor growth compared with wild type in vivo. In contrast, STEAP-1 introduced tumor cells augmented the tumor growth compared with STEAP-1-negative wild type cells. Using dye transfer assay, we demonstrate that the STEAP-1 is involved in intercellular communication between tumor cells and adjacent tumor stromal cells and therefore may play a key role for the tumor growth in vivo. These data indicate the inhibition of the STEAP-1 function or expression can be a new strategy for cancer therapy.

DOI： 10.1016/j.yexcr.2013.07.025

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1(br)) cells by the ALDEFLUOR assay. Small proline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1(br) cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1(br) cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4.

DOI： 10.1016/j.bbrc.2013.08.021

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We have shown that ECRG4 suppressed Fas-induced apoptosis in Jurkat cells. ECRG4 mRNA expression was ubiquitously detected in normal adult human tissues, suggesting that ECRG4 plays a major role in human tissues. ECRG4 mRNA expression was down-regulated in tumor cells. Expression of ECRG4 suppressed cell growth. We established an anti-ECRG4 monoclonal antibody. Our immunohistochemical analysis demonstrated that ECRG4-positive cells tended to be distributed in the region that was negative for Ki-67 in esophageal squamous cell carcinoma tissues. There was a significant inverse correlation between ECRG4 expression and Ki-67 labeling index in esophageal squamous cell carcinoma. This study provides the first functional evidence for an association of endogenous expression of ECRG4 with cell proliferation. ECRG4 is a candidate tumor suppressor gene that might be involved in the proliferation of esophageal squamous cell carcinoma.

DOI： 10.1111/pin.12079

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/tumour-initiating cells (TICs) are defined as the small population of cancer cells that have stem cell-like phenotypes and high capacity for tumour initiation. These cells may have a huge impact in the field of cancer therapy since they are extremely resistant to standard chemoradiotherapy and thus are likely to be responsible for disease recurrence after therapy. Therefore, extensive efforts are being made to elucidate the pathological and molecular properties of CSCs/TICs and, with this information, to establish efficient anti-CSC/TIC targeting therapies. This review considers recent findings on stress response genes that are preferentially expressed in CSCs/TICs and their roles in tumour-promoting properties. Implications for a novel therapeutic strategy targeting CSCs/TICs are also discussed.

DOI： 10.3109/02656736.2013.814809

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously, we showed that the expression of potassium channel tetramerization domain-containing 12 (KCTD12), which was discovered by a proteomics approach, is associated with high-risk behavior of gastrointestinal stromal tumors (GISTs). Here, we examined the distribution and expression of this protein by immunostaining with a commercially available polyclonal KCTD12 antibody in GISTs (n = 64) and other types of malignancy (n = 168) to clarify its diagnostic and clinical significance. Diffuse KCTD12 immunoreactivity was found in most GISTs (52 cases; 81%). KCTD12 expression was observed primarily in vascular endothelial cells, Purkinje cells of the cerebellum, and some neurons scattered throughout the cerebral cortex. KCTD12 was absent from not only the interstitial cells of Cajal but also interstitial cells of Cajal hyperplasia that was encountered incidentally in colon diverticulitis. KCTD12 immunostaining was also seen in malignant peripheral nerve sheath tumors (2/10 cases; 20%), synovial sarcomas (2/10; 20%), solitary fibrous tumor (1/8; 13%), angiosarcoma (1/7; 14%), and colon adenocarcinoma (1/24; 4%). In survival analyses, the 5-year recurrence-free survival rate of patients without KCTD12 expression was only 16.7% compared with 95.6% in those with KCTD12 expression (P < .0001). Ki-67 and KCTD12 were significant predictors of recurrence-free survival, and KCTD12 expression provided additional information about recurrence-free survival after accounting for Ki-67 status. Overall, KCTD12 expression was specific for GISTs from neoplastic and nonneoplastic adult tissues other than brain and served as a predictor of GIST recurrence. These findings suggest that KCTD12 is a useful and reliable biomarker for both the diagnosis and prognosis of GIST.

DOI： 10.1016/j.humpath.2012.10.013

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記述言語：英語  

DOI： 10.2217/imt.13.44

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.

DOI： 10.1016/j.yexmp.2012.10.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Prostate cancer cells include a small population of cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) that have roles in initiation and progression of the cancer. Recently, we isolated prostate CSCs/CICs as aldehyde dehydrogenase 1-highh (ALDH1(high) ) cells using the ALDEFLUOR assay; however, the molecular mechanisms of prostate CSCs/CICs are still elusive. Prostate CSCs/CICs were isolated as ALDH1(high) cells using the ALDEFLUOR assay, and the gene expression profiles were analyzed using a cDNA microarray and RT-PCR. We found that prostate CSCs/CICs expressed higher levels of growth factors including hepatocyte growth factor (HGF). Hepatocyte growth factor protein expression was confirmed by enzyme linked immunosorbent assay and Western blotting. On the other hand, c-MET HGF receptor was expressed in both CSCs/CICs and non-CSCs/CICs at similar levels. Hepatocyte growth factor and the supernatant of myofibloblasts derived from the prostate augmented prostasphere formation in vitro, and prostasphere formation was inhibited by an anti-HGF antibody. Furthermore, c-MET gene knockdown by siRNA inhibited the prostasphere-forming ability in vitro and tumor-initiating ability in vivo. Taken together, the results indicate that HGF secreted by prostate CSCs/CICs and prostate myofibroblasts has a role in the maintenance of prostate CSCs/CICs in an autocrine and paracrine fashion.

DOI： 10.1111/cas.12104

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin, a member of the inhibitor of apoptosis protein gene family, inhibits apoptosis and promotes mitosis. We determined whether nuclear or cytoplasmic localization of survivin could predict survival of patients with upper urinary tract urothelial carcinoma (UUTUC). Immunohistochemical staining for survivin was carried out on archival specimens from 125 consecutive patients with UUTUC who underwent radical nephroureterectomy. Nuclear and cytoplasmic staining of survivin was scored and compared with clinicopathologic features and cancer-specific survival (CSS). Nuclear expression of survivin was significantly correlated with tumor grade (p < 0.001), lymphovascular invasion (p = 0.022) and poor survival with an estimated 5-year CSS probability of 54 % for tumors with nuclear expression of survivin vs. 73 % for those without nuclear expression of survivin (hazard ratio = 2.19; 95 % confidence interval = 1.02-4.70; p = 0.043). The 5-year cancer-specific survival rates of patients with cytoplasmic survivin-negative and -positive tumors were 66 and 67 %, respectively. There was no difference in survival between patients with cytoplasmic survivin-negative tumors and those with cytoplasmic survivin-positive tumors. Using univariate analysis, nuclear survivin expression, tumor grade, pathological T stage, pathological N stage, and lymphovascular invasion were the predictive variables for CSS. In contrast, cytoplasmic survivin expression had no prognostic relevance. These data suggest that nuclear accumulation of survivin represents biologic aggressiveness and that nuclear survivin is a negative prognostic marker in patients with resected UUTUC.

DOI： 10.1007/s00428-012-1343-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aldehyde dehydrogenase 1 (ALDH1) and sex determining region-Y-related high mobility group box 2 (SOX2) have been identified as putative cancer stem-like cell/tumor-initiating cell markers in various cancer tissues. The aim of this study was to elucidate the prognostic impact of these putative cancer stem-like cell/tumor-initiating cell markers in upper urinary tract urothelial cell carcinoma. Immunohistochemical staining for ALDH1 and SOX2 was carried out on archival specimens from 125 patients with upper urinary tract urothelial cell carcinoma who underwent radical nephroureterectomy. The prognostic value of ALDH1 and SOX2 expression and other clinicopathological features was evaluated. On univariate analysis, tumor grade, pathological T stage, pathological N stage, lymphovascular invasion, ALDH1 expression and SOX2 expression were associated with a poor prognosis. On multivariate analysis, the independent factors of prognosis were tumor grade (P=0.014), pathological N stage (P=0.005) and ALDH1 expression (P=0.002). In subgroup analysis, those subgroups with no positive, one positive or two positive results in immunohistochemistry for ALDH1 and SOX2 expression had estimated 5-year cancer-specific survival rates of 80%, 49% and 22%, respectively (P<0.001). Neither ALDH1 nor SOX2 expression correlated with intravesical recurrence after radical nephroureterectomy. These findings suggest that cancer stem-like cells/tumor-initiating cells are linked to more aggressive behavior of upper urinary tract urothelial cell carcinoma, supporting the current cancer stem cell hypothesis. Thus, therapeutic targeting of cancer stem-like cells/tumor-initiating cells in upper urinary tract urothelial cell carcinoma is a future possibility.

DOI： 10.1038/modpathol.2012.139

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin-2B80-88 plus incomplete Freund's adjuvant (IFA) and α-interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme-linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin-2B80-88 peptide-specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin-2B80-88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor-prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905.

DOI： 10.1111/cas.12046

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have self-renewal ability, differentiation ability and high tumor-initiating ability. CSCs/CICs are resistant to cancer therapies including chemotherapy and radiotherapy. Therefore, CSCs/CICs are thought to be responsible for cancer recurrence and distant metastasis after treatment. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we isolated CSCs/CICs as side population (SP) cells from lung carcinoma, colon carcinoma and breast carcinoma cells and analyzed the molecular mechanisms of CSCs/CICs. cDNA micro-array screening and RT-PCR analysis revealed that sperm mitochondria-associated cysteine-rich protein (SMCP) is ectopically expressed in SP cells. 5'-Rapid amplification of cDNA end (RACE) analysis revealed that the SMCP transcript in SP cells was a variant form (termed vt2) which is composed from only one exon. SMCP vt2 was detected in only cancer cells, whereas the wild-type (vt1) form of SMCP was expressed in the testis. SMCP was shown to have a role in tumor initiation by SMCP overexpression and SMCP knockdown using siRNAs in lung cancer cells. Taken together, the initiation results indicate that an ectopically expressed variant form of SMCP has a role in tumor initiation of CSCs/CICs and that the variant form of SMCP might be a novel CSC/CIC marker and a potential and promising target of CSC/CIC-targeting therapy.

DOI： 10.1371/journal.pone.0069095

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have high tumorigenicity. Furthermore, CSCs/CICs are resistant to several cancer therapies, and CSCs/CICs are therefore thought to be responsible for cancer recurrence after treatment and distant metastasis. In epithelial ovarian cancer (EOC) cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian CSCs/CICs are involved. There are four major histological subtypes in EOC, and serous adenocarcinoma and clear cell adenocarcinoma are high-grade malignancies. We therefore analyzed ovarian CSCs/CICs from ovarian carcinoma cell lines (serous adenocarcinoma and clear cell adenocarcinoma) and primary ovarian cancer cells in this study. We isolated ovarian CSCs/CICs as an aldehyde dehydrogenase 1 high (ALDH1(high)) population from 6 EOC cell lines (3 serous adenocarcinomas and 3 clear cell adenocarcinomas) by the ALDEFLUOR assay. ALDH1(high) cells showed greater sphere-forming ability, higher tumorigenicity and greater invasive capability, indicating that ovarian CSCs/CICs are enriched in ALDH1(high) cells. ALDH1(high) cells could also be isolated from 8 of 11 primary ovarian carcinoma samples. Immunohistochemical staining revealed that higher ALDH1 expression levels in ovary cancer cases are related to poorer prognosis in both serous adenocarcinoma cases and clear cell adenocarcinoma cases. Taken together, the results indicate that ALDH1 is a marker for ovarian CSCs/CICs and that the expression level of ALDH1 might be a novel biomarker for prediction of poor prognosis.

DOI： 10.1371/journal.pone.0065158

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, a member of the inhibitor of apoptosis protein family, recognized by CD8+cytotoxic T lymphocytes (CTL). In a phase I clinical trial of survivin-2B80-88 vaccination for metastatic urothelial cancer (MUC), we achieved clinical and immunological responses with safety. Moreover, our previous study indicated that interferon alpha (IFN α ) enhanced the effects of the vaccine for colorectal cancer. Therefore, we started a new phase I clinical trial of survivin-2B80-88 vaccination with IFN α for MUC patients. Twenty-one patients were enrolled and no severe adverse event was observed. HLA-A24/survivin-2B80-88 tetramer analysis and ELISPOT assay revealed a significant increase in the frequency of the peptide-specific CTLs after vaccination in nine patients. Six patients had stable disease. The effects of IFN α on the vaccination were unclear for MUC. Throughout two trials, 30 MUO patients received survivin-2B80-88 vaccination. Patients receiving the vaccination had significantly better overall survival than a comparable control group of MUO patients without vaccination (P = 0.0009). Survivin-2B80-88 vaccination may be a promising therapy for selected patients with MUC refractory to standard chemotherapy. This trial was registered with UMIN00005859.

DOI： 10.1155/2013/262967

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH(Br)) cells from ovarian cancer cells. Both SP cells and ALDH(Br) cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH(Low) cells, respectively. We analyzed an SP and ALDH(Br) overlapping population (SP/ALDH(Br)), and the SP/ALDH(Br) population exhibited higher tumor-initiating ability than that of SP cells or ALDH(Br) cells, enabling initiation of tumor with as few as 10(2) cells. Furthermore, SP/ADLH(Br) population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH(Low), MP/ALDH(Br) and MP/ALDH(Low) cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH(Br) population was detected in several gynecological cancer cells with ratios of 0.1% for HEC-1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH(Br) overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.

DOI： 10.1371/journal.pone.0068187

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記述言語：英語  

DOI： 10.1111/pin.12017

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients' peripheral blood, and the CTL activities were investigated in several temperature conditions (37-41 °C). Cytotoxicity and IFN-γ secretion of CTL were greatest under 39 °C condition, whereas they were minimum under 41 °C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCRαβ by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 °C. On the other hand, CTL cell death was induced under 41 °C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 °C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

DOI： 10.1007/s12192-012-0348-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Evaluation of: Rech AJ, Mick R, Martin S et al. CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patents. Sci. Transl. Med. 4(134), 134ra62 (2012). Tregs are involved in the maintenance of immunological self-tolerance. Recent studies have revealed that Tregs suppress antitumor immunity and that they are major obstacles for cancer immunotherapy. Various approaches have been carried out to cancel immunological suppression by Tregs in clinical settings; however, side effects such as autoimmunity occurred and expected antitumor effects were not achieved. In a recent study, Rech et al. evaluated daclizumab, a US FDA-approved humanized anti-CD25 antibody, for regulation of Treg cells in a peptide vaccination trial of breast cancer patients. Daclizumab caused long-lasting depletion of CD25(+) Tregs, reprogramming of CD25(+) Tregs, and enhancement of antipeptide immune response. Of note, major autoimmune responses were not observed in daclizumab-treated patients. This study provides a possible safe and promising approach to regulate Tregs in cancer vaccine therapy.

DOI： 10.2217/imt.12.116

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

One of the major factors involved in the prognosis of oral squamous cell carcinoma (OSCC) patients is metastasis. Recent progress in cancer stem-like cell/cancer-initiating cell (CSC/CIC) research indicates that CSCs are related to metastasis. Aldehyde dehydrogenase 1 - (ALDH1) and SRY-related HMG-box gene 2 (SOX2) have recently been shown to be putative CSC markers for several human malignancies. The aim of this study was to determine the association of ALDH1 and SOX2 expression in oral squamous cell carcinoma (OSCC) with lymph node metastasis. Immunohistochemical staining of ALDH1, SOX2 and Ki67 was performed in 80 OSCC tissues. High expression rates of ALDH1 (2%-40%) were found to be related to lymph node metastasis (P = 0.0017). Interestingly, we found that SOX2 staining could be classified into two patterns: (i) peripheral staining pattern; and (ii) diffuse staining pattern. The diffuse staining pattern showed a significant correlation with lymph node metastasis (P < 0.001). No correlation was found between Ki67 staining and lymph node metastasis (P = 0.4724). The ALDH1 positive staining rates in metastatic lymph nodes were higher than that in corresponding primary OSCC tissues. These results indicate that high expression rates of ALDH1 and SOX2 diffuse staining patterns might be novel prediction markers for OSCC lymph node metastasis.

DOI： 10.1111/j.1440-1827.2012.02851.x

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells, which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. MATERIALS AND METHODS: The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. RESULTS: ALDH1(high) cells were detected in 6.8% of 22Rv1 cells, which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p < 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. CONCLUSIONS: Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay.

DOI： 10.1016/j.juro.2012.02.2555

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC.

DOI： 10.1158/0008-5472.CAN-11-3062

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8-5, was established. It could detect HLA-A, -B, and -C antigens in formalin-fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8-5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2-microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down-regulation might be a common characteristic of breast cancer mostly caused by the down-regulation of beta2-microglobulin expression.

DOI： 10.1111/j.1440-1827.2012.02789.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

DOI： 10.1093/carcin/bgs118

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The aim of this study was to establish a novel efficient cancer DNA vaccine approach. Many tumor-associated antigens (TAAs) have been reported; however, there is little information of the efficiency of each TAA. Normal cells barely undergo mitosis, whereas cancer cells divide frequently and grow well. Thus, G2/M-related antigens are cancer cell-specific and are regarded to be suitable candidates as targets of cancer immunotherapy. In this study, we compared the efficiencies of G2/M-related antigens including Birc5, Aurka, Nke2 and Plk1 by using a DNA vaccination model. Mice that had been immunized with G2/M-related antigens coding plasmid were challenged with CT26 colon cancer cells. Interestingly, Birc5- and Aurka-immunized mice showed an anti-tumor effect, whereas Nek2- and Plk1-immunized mice did not show any anti-tumor effect. We investigated the expression of G2/M-related antigens in cancer stem-like cell (CSC)/cancer-initiating cell (CIC) population to verify the difference in the anti-tumor effect. CSCs/CICs were isolated as side population (SP) cells using Hoechst 33342 dye from CT 26 cells. It was found that Birc5 and Aurka are expressed in both CSCs/CICs and non-CSCs/CICs (shared antigens), whereas Nek2 and Plk1 are expressed preferentially in non-CSCs/CICs (non-CSC antigens). Therefore, antigen expression in the CSC/CIC population might be related to the anti-tumor efficiency of cancer immunotherapy. Furthermore, we established a heat shock protein (Hsp90)-fused Birc5 plasmid to improve anti-cancer immunity. Birc5 fused to the N-terminal region of Hsp90 showed a stronger anti-tumor effect, whereas Birc5 fused to the C-terminal region of Hsp90 did not show enhancement compared with Birc5. These observations indicate that expression in the CSC/CIC population is essential to achieve tumor regression and that fusing antigens to the N-terminal region of Hsp90 enhances the anti-tumor effect.

DOI： 10.1016/j.yexmp.2011.09.016

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The aim of this study was to establish an efficient human papilloma virus (HPV) type 16-targeting cancer immunotherapy. Persistent high-risk HPV infection causes cervical intra-epithelial neoplasia (CIN) and subsequent cervical carcinoma. HPV type16 (HPV16) is one of the common carcinogenic types and is found in about 50% of invasive cervical carcinomas. HPV16-derived viral proteins E6 and E7 are expressed in cancerous cells through the progression of the disease and have a role in carcinogenesis but are not expressed in normal cells. Thus, these proteins are regarded as ideal antigens for cervical carcinoma immunotherapy. In this study, we generated a novel HPV 16 E6 and E7 gene plasmid containing oligomannose liposomes (OML-HPV). We compared the cytotoxic T lymphocyte (CTL) induction efficiency of OML-HPV and that of standard liposome-HPV16 E6 and E7 DNA complex. HPV16 E6-specific CTLs could be generated from HPV 16-positive cervical carcinoma patient's peripheral blood mononuclear cells (PBMCs) by stimulating OML-HPV, but could not by stimulating standard liposome-HPV 16 E6, E7 DNA complex. Furthermore, we screened HLA-A24-restricted HPV16 E6- and E7-derived peptides, and found that one E6-derived peptide (E6 66-74) showed the highest immunogenicity with ELISPOT assay from 100% of HPV16-positive patients (4 out of 4). On the other hand, other E6- or E7-derived peptides, including E6 49-57, E6 82-90, E6 87-95, E6 98-106 and E7 83-93, showed less frequent reactivity. These results indicate that OML-HPV is a more effective approach than DNA vaccination using standard liposomes, and that a novel HLA-A24-restricted peptide, E6 66-74, might be a suitable target of cervical cancer immunotherapy.

DOI： 10.1016/j.yexmp.2011.10.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem cells (CSCs)/cancer-initiating cells (CICs) are characterized as a small population of cancer cells that have high tumor-initiating ability. CSCs/CICs are resistant to several cancer therapies, and eradication of CSCs/CICs is essential to cure cancer. How can we eradicate CSCs/CICs? Cytotoxic T lymphocytes (CTLs) might be a promising answer.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recently, the SOX2 gene has been reported to be amplified in human lung squamous cell carcinomas. However, its roles in human lung adenocarcinomas are still elusive. In this study, we analyzed the functions of SOX2 in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) derived from human lung adenocarcinoma. Human lung CSCs/CICs were isolated as higher tumorigenic side population (SP) cells using Hoechst 33342 dye from several lung cancer cell lines. Four of nine lung cancer cell lines were positive for SP cells (LHK2, 1-87, A549, Lc817). The ratios of SP cells ranged from 0.4% for Lc817 to 2.8% for LHK2. To analyze the molecular aspects of SP cells, we performed microarray screening and RT-PCR analysis, and isolated SOX2 as one of a SP cell-specific gene. SOX2 was expressed predominantly in LHK2 and 1-87 SP cells, and was also expressed in several other cancer cell lines. The expression of SOX2 protein in primary human lung cancer tissues were also confirmed by immunohistochemical staining, and SOX2 was detected in more than 80% of primary lung cancer tissues. To address SOX2 molecular functions, we established a SOX2-overexpressed LHK2 and A549 cell line (LHK2-SOX2 and A549-SOX2). LHK2-SOX2 cells showed higher rates of SP cells and higher expression of POU5F1 compared with control cells. LHK2-SOX2 and A549-SOX2 cells showed relatively higher tumorigenicity than control cells. On the other hand, SOX2 mRNA knockdown of LHK2 SP cells by gene-specific siRNA completely abrogated tumorigenicity in vivo. These observations indicate that SOX2 has a role in maintenance of stemness and tumorigenicity of human lung adenocarcinoma CSCs/CICs and is a potential target for treatment.

DOI： 10.1038/labinvest.2011.140

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lengsin is an eye lens protein with a glutamine synthetase domain. We previously identified this protein as a lung carcinoma antigen through cDNA microarray analysis. Lengsin protein is overexpressed irrespective of the histological type of lung carcinoma, but not in normal tissues other than the lens. Therefore, to significantly extend the use of Lengsin-based T-cell immunotherapies for the treatment of patients with lung carcinoma, we searched for HLA-A*0201-restricted epitopes from this protein by screening predicted Lengsin-derived candidate peptides for the induction of tumor-reactive CTLs. Four Lengsin-derived peptides were selected by computerized algorithm based on a permissive HLA-A*0201 binding motif, and were used to immunize HLA-A*0201 transgenic (HHD) mice. Two of the immunizing peptides, Lengsin(206-215)(FIYDFCIFGV) and Lengsin(270-279)(FLPEFGISSA), induced peptide-specific cytotoxic T lymphocytes (CTLs) in HHD mice, and thus were used to stimulate human peripheral blood lymphocytes in vitro. Lengsin(206-215) and Lengsin (270-279) also induced human peptide-specific CTLs, and we were able to generate Lengsin(206-215)- and Lengsin(270-279)-specific CTL clones. The Lengsin(270-279)-specific CTL clone specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and Lengsin, and HLA-A*0201+/Lengsin+ lung carcinoma cells in an HLA-A*0201-restricted manner. On the other hand, the Lengsin(206-215)-specific CTL clone failed to recognize HLA-A*0201+/Lengsin+ target cells in the absence of cognate peptide. These results suggest that Lengsin(270-279) is naturally processed and presented by HLA-A*0201 molecules on the surface of lung carcinoma cells and may be a new target for antigen-specific T-cell immunotherapy against lung cancer.

DOI： 10.3892/ijo.2011.1089

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Evaluation of: Watkins SK, Zhu Z, Riboldi E et al. FOXO3 programs tumor-associated DCs to become tolerogenic in human and murine prostate cancer. J. Clin. Invest. 121(4), 1361-1372 (2011). Tumor-associated dendritic cells (TADCs) have been described as immune-suppressive cells in cancers, and part of the molecular mechanisms has emerged. The transcription factor FOXO3--one of the tumor suppressors--is overexpressed in TADCs that have been infiltrated in human prostate cancers and TRAMP mouse model of prostate cancers, and induces the expression of immune-suppressive genes including indoleamine-2,3-dioxygenase (IDO1), arginase (ARG1) and TGF-β. Adoptive transfer of T helper cells or silencing of FOXO3 by siRNAs repressed the expression of FOXO3 gene and inhibited the tolerogenicity of TADCs. Therefore, inhibition of FOXO3 signals might be a clue for improvement of cancer immunotherapy.

DOI： 10.2217/imt.11.94

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin-2B80-88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin-2B80-88 plus incomplete Freund's adjuvant (IFA); and (ii) survivin-2B80-88 plus IFA and a type-I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin-2B80-88 plus IFA was not significantly different from that with survivin-2B80-88 alone, treatment with the vaccination protocol of survivin-2B80-88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin-2B80-88 peptide-specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme-linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single-cell clone separation by cell sorting of peptide-specific CTL showed that each CTL clone was indeed not only peptide-specific but also cytotoxic against human cancer cells in the context of the expression of both HLA-A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin-2B80-88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers.

DOI： 10.1111/j.1349-7006.2011.01918.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: To verify the efficacy of a serum autoantibody against hypoxia-inducible factor prolyl hydroxylase-3 (PHD3) as a serological marker for RCC. METHODS: Serum samples and surgically resected tumor tissue specimens were obtained from 22 patients with primary RCC, 15 of whom underwent radical nephrectomy and 7 partial nephrectomy. Preoperative serum samples were obtained just before tumor resection. Postoperative serum samples were obtained from 17 patients at least 1 month after tumor removal. Serum samples were also obtained from 26 healthy volunteers. Titers of the anti-PHD3 antibody (Ab) were determined by enzyme-linked immunosorbent assay. RESULTS: Serum anti-PHD3 Ab titers were significantly higher in patients with RCC than in healthy volunteers (0.610 ± 0.023 vs. 0.591 ± 0.031, P = 0.0001). Using a cutoff point of 0.599, sensitivity, specificity, and positivity for prediction of RCC were 86.4, 57.7, and 63.3%, respectively. In all 17 patients, titers of serum anti-PHD3 were decreased after the surgical resection compared with those before operation (0.622 ± 0.023 vs. 0.580 ± 0.024, P = 0.0003). CONCLUSIONS: The present study suggests that the anti-PHD3 Ab may be a novel serological marker for RCC and the titer may reflect the tumor burden in each individual.

DOI： 10.1007/s00432-010-0940-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8(+) T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8(+) T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag-BiP complex.

DOI： 10.4049/jimmunol.1004048

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs.

DOI： 10.1016/j.ajpath.2011.01.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is abundantly expressed in most malignancies, but is hardly detectable in normal adult tissues. Previously we have identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) cytotoxic T lymphocytes (CTL). Survivin-2B80-88-specific CTL were induced efficiently from peripheral blood mononuclear cells (PBMC) of oral cancer patients after stimulation with the peptide in vitro. We conducted a phase I clinical study to evaluate the safety and the efficacy of survivin-2B80-88 peptide vaccination in HLA-A24-positive patients with advanced or recurrent oral cancer. The vaccines were given subcutaneously or intratumorally six times at 14-day intervals. Eleven patients were enrolled and 10 patients completed the vaccination protocol. No adverse events were observed in any patients. In two patients, the levels of serum squamous cell carcinoma (SCC) antigen decreased transiently during the period of vaccination. Tumor regression that was compatible with a partial response (PR) was noted in one patient. The remaining nine patients experienced progressive disease (PD). Immunologically, an increase of the peptide-specific CTL frequency was detected in six of the eight patients evaluated by HLA-A24/peptide tetramer analysis. The present clinical trial revealed that survivin-2B peptide vaccination was safe and had therapeutic potential for oral cancer patients. However, subsequent clinical trials in combination with various adjuvant drugs will be required to improve the immunological and therapeutic efficacy. This trial was registered with University Hospital Medical Information Network (UMIN) number UMIN000000976.

DOI： 10.1111/j.1349-7006.2010.01789.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In our previous study, we demonstrated that a peptide derived from the novel centrosome residing protein Cep55/c10orf3 can be targeted by the cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of breast carcinoma patients. In this report, we evaluated the feasibility of cancer immunotherapy using Cep55/c10orf3 peptide for colorectal carcinoma (CRC). To evaluate the expression of Cep55/c10orf3 in CRC tissues, we performed immunohistochemical staining of using anti-Cep55/c10orf3 monoclonal antibody. Sixty-three percent cases showed weak positive for Cep55/c10orf3 in total 70 CRC cases. The Cep55/c10orf3 expression intention was collated with high histological grade of CRC. Thus, we hypothesized that Cep55/c10orf3 can also be the target of CTLs in CRC cases. We generated CTLs from PBMCs of human leukocyte antigen (HLA)-A24-positive colorectal carcinoma patients using HLA-A24-restricted Cep55/c10orf3 peptides. Two of 6 colorectal cancer patients were reactive for the Cep55/c10orf3_193(10) peptide, which was the only immunogenic peptide in breast carcinoma patients. CTL clone specific for Cep55/c10orf3_193(10) recognized and lysed HLA-A24 (+) and Cep55/c10orf3 (+) colorectal carcinoma cell lines. In addition, 1 of 6 colorectal carcinoma patients was reactive for the Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) peptides, but not for Cep55/c10orf3_193(10) with the ELISPOT assay. These observations suggest that the antigenic peptide repertoire presented by HLA-A24 in colorectal carcinoma might be different from that in breast carcinoma. Thus, these peptide vaccination peptide mixture of Cep55/c10orf3_193(10), Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) might be more effective than a single peptide in the treatment of colorectal carcinoma patients.

DOI： 10.1016/j.yexmp.2010.10.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human leukocyte antigen (HLA) typing is essential to carry out HLA-class I restricted antigenic peptide-based cancer immunotherapy. To establish a one-step polymerase chain reaction-sequence-specific primer (PCR-SSP) method, we designed two novel HLA-A24-specific primer sets and determined the optimal conditions for specific amplification. Then, we performed HLA-A24 typing of two healthy donors' and 17 cancer patients' peripheral blood with serological typing and PCR-SSP typing. Eleven of the 19 cases were determined HLA-A24-positive by the PCR-SSP method precisely; however, five cases showed false positive with serological analysis. Thus, for HLA-A24 typing in the Japanese population, the PCR-SSP method is faster and more accurate than serological typing.

DOI： 10.1080/15321819.2010.543219

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2-Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.

DOI： 10.1016/j.yexmp.2009.12.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer stem-like cells (CSCs)/tumor-initiating cells (TICs) are a small population of cancer cells that have the properties of tumor-initiating ability, self-renewal and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence and distant metastasis. Thus, elimination of CSCs/TICs is essential to cure malignant diseases. However, there are several studies reporting that CSCs/TICs are more resistant to standard cancer therapies, including chemotherapy and radiotherapy, than non-CSC/TIC populations. How then, can we eliminate CSCs/TICs? Immunotherapy might be the possible answer. In recent analysis, innate immunity (natural killer cells and gammadeltaT cells) and also adaptive immunity (cytotoxic T lymphocyte-based cellular immunity and antibody-based humoral immunity) can recognize CSCs/TICs in vitro efficiently. Furthermore, CSC/TIC-specific monoclonal antibody therapies are also efficient in vivo. In this article, we describe the potency, possibilities and problems of CSC/TIC-targeting immunotherapy.

DOI： 10.2217/imt.10.10

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.

DOI： 10.1016/j.yexmp.2009.09.021

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Alpha-methylacyl-CoA racemase (AMACR) is an enzyme playing an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives. High expression levels of AMACR have been described in various cancers, including prostate cancer, colorectal cancer and kidney cancer. Because of its cancer-specific and frequent expression, AMACR could be an attractive target for cytotoxic T-lymphocyte (CTL)-based immunotherapy for cancer. In the present study, we examined the induction of AMACR-specific CTLs from prostate cancer patients' peripheral blood mononuclear cells (PBMCs) and determined HLA-A24-restricted CTL epitopes.RT-PCR and immunohistochemical analysis revealed that AMACR was strongly expressed in prostate cancer cell lines and tissues as compared with benign or normal prostate tissues. Four AMACR-derived peptides carrying the HLA-A24-binding motif were synthesized from the amino acid sequence of this protein and analyzed to determine their binding affinities to HLA-A24. By stimulating patient's PBMCs with the peptides, specific CTLs were successfully induced in 6 of 11 patients. The peptide-specific CTLs exerted significant cytotoxic activity against AMACR-expressing prostate cancer cells in the context of HLA-A24. Our study demonstrates that AMACR could become a target antigen for prostate cancer immunotherapy, and that the AMACR-derived peptides might be good peptide vaccine candidates for HLA-A24-positive AMACR-expressing cancer patients.

DOI： 10.1186/1479-5876-7-103

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Using a novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody (EMR 8-5) reacting with paraffin-embedded sections, we examined the prognostic significance of HLA class I molecules in muscle-invasive bladder cancer patients who underwent radical cystectomy. Immunohistochemical staining for HLA class I molecules with monoclonal antibody EMR 8-5 was performed on specimens from 65 clinically muscle-invasive bladder cancer patients who underwent radical cystectomy and pelvic lymph node dissection without neoadjuvant chemotherapy. We analyzed the clinicopathological and prognostic significance of HLA class I expression. Immunohistochemical analysis revealed HLA class I down-regulation in 22 (33.8%) invasive bladder cancers. This down-regulation had no correlation with clinicopathological parameters such as pathologic stage, nodal status, and grade. The recurrence-free survival of patients with HLA class I-positive tumors was significantly better than that of those with down-regulation (log rank, P = 0.0337). Multivariate analysis revealed that HLA class I expression was a significant factor influencing the recurrence-free survival of bladder cancer patients after cystectomy (P = 0.0155). Our data demonstrate that HLA class I down-regulation in tumor cells was clearly observed in about one-third of the patients. HLA class I expression could be a prognostic marker for muscle-invasive bladder cancer patients after cystectomy.

DOI： 10.1111/j.1349-7006.2009.01329.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit intriguing, efficient CTL responses by cross-presentation via an as yet entirely unknown mechanism. Oxygen-regulated protein 150 (ORP150), also known as grp170, is an endoplasmic reticulum-resident HSP and is up-regulated by hypoxia. It has been demonstrated that ORP150 binds tumor-associated Ag peptides within cancer cells. Immunization with an ORP150-tumor Ag complex has been shown to generate tumor-specific CTLs. Most recently, it has been shown that exogenous ORP150 induces cross-presentation of a chaperoned Ag, thereby stimulating Ag-specific CTLs. However, the mechanism underlying this efficient cross-presentation is still unsolved. In this study, we show that the ORP150-precursor peptide complex can elicit CTL response through cross-presentation as well as the CD4(+) T cell response by dendritic cells. Furthermore, we observed that the internalized ORP150-peptide complex, but not OVA protein, which was not cross-presented, was sorted to the Rab5(+), EEA1(+) static early endosome, followed by translocation to a recycling endosome, where the ORP150-chaperoned peptide was processed and bound to MHC class I molecules. Moreover, we observed that immunization of mice with ORP150-peptide complexes elicited strong peptide-specific CTLs and antitumor effects in vivo. Our data indicate that targeting of the Ag to a "static" early endosme by ORP150 is required for the efficient cross-presentation.

DOI： 10.4049/jimmunol.0803768

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in many malignant tumors including urothelial cancer but is hardly detectable in normal, differentiated adult tissues. Previously we reported CD8-positive cytotoxic T-lymphocytes (CTLs) were successfully induced by stimulation with survivin-2B80-88 peptide in vitro. We started a phase I clinical study of survivin-2B80-88 peptide vaccination for advanced urothelial cancer patients to assess the safety and efficacy of this vaccination. Nine patients were received vaccination and were evaluated for immunological evaluation, adverse events, and clinical responses. A total of 46 vaccinations were carried out. There was no severe adverse event. HLA-A24/survivin-2B80-88 peptide tetramer analysis revealed a significant increase in the peptide-specific CTL frequency after the vaccination in five patients. Slight reduction of the tumor volume was observed in one patient. Survivin-2B80-88 peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in urothelial cancer patients.

DOI： 10.1007/s00262-009-0691-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A glutamine synthetase I family protein, Lengsin, was previously identified as a novel lens-specific transcript in the vertebrate eye. In this report, we show for the first time that Lengsin is a novel tumor-associated antigen expressed ectopically in lung cancer. Interestingly, a novel spliced form of human Lengsin termed 'splicing variant 4', gaining exon 3 that codes extra 63 amino acids, is the dominant transcript form in lung cancer cells. Lengsin mRNA could be detected in 7 of 12 (58%) lung cancer cell lines and 7 of 7 (100%) surgically resected lung cancer tissues. On the other hand, Lengsin transcripts could not be detected in normal major tissues or in other cancer cell lines, including melanoma, colorectal carcinoma, breast carcinoma and hepatocellular carcinoma. In addition, knockdown of Lengsin mRNA with RNAi caused cell death and a decrease of cell viability, suggesting that Lengsin has some essential role in cell survival. Since the lens is an immune-privileged site, we regard Lengsin as a highly immunogenic cancer antigen. Anti-Lengsin autoantibodies were detectable in sera of lung cancer patients, although these patients did not show any lens-related disturbances. Hence, Lengsin splicing variant 4 might be an immunogenic lung cancer-specific antigen that is suitable as a diagnostic marker and for molecular targeting therapy, including immunotherapy.

DOI： 10.1111/j.1349-7006.2009.01187.x

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記述言語：英語  

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

DOI： 10.1186/1479-5876-7-45

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Identification of tumor-associated antigens may facilitate vaccination strategies to treat patients with malignant diseases. We have found that the centrosomal protein, Cep55/c10orf3 acts as a novel breast carcinoma-associated tumor-associated antigen. Cep55/c10orf3 mRNA was detectable in a wide variety of tumor cell lines. Expression was barely detectable in normal tissues except for testis and thymus. Moreover, Cep55/c10orf3 protein could be detected by a monoclonal anti-Cep55/c10orf3 antibody (# 11-55) in 69.8% of breast carcinoma, 25% of colorectal carcinoma, and 57.8% of lung carcinoma tissues. The expression of Cep55/c10orf3 protein did not show any relationship with the hormone receptors such as estrogen receptor and progesterone receptor or expression patterns of p185 HER2/neu. We designed 11 peptides which displayed a human leukocyte antigen-A24 binding motif. One Cep55/c10orf3-peptide, Cep55/c10orf3_193(10) (VYVKGLLAKI), induced cytotoxic T lymphocytes (CTLs) in 3 of 3 patients with Cep55/c10orf3 (# 11-55)-positive breast carcinoma. A Cep55/c10orf3_193(10)-specific CTL clone could also recognize Cep55/c10orf3 (+) displayed on human leukocyte antigen-A24 (+) cancer cell lines. These data indicate that Cep55/c10orf3 peptides were naturally presented by breast cancer cells and can cause CTL clonal expansion in vivo. Monoclonal antibody # 11-55 and the Cep55/c10orf3_193(10) peptides may be useful as part of a therapeutic strategy for hormonal therapy or anti-p185 HER2/neu monoclonal antibody therapy-resistant breast carcinoma patients.

DOI： 10.1097/CJI.0b013e3181a1d109

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Antigenic peptides derived from tumor-associated antigens (TAAs) facilitate peptide cancer vaccine therapies. With the recent progress in cancer immunity research, huge amounts of antigenic peptides have already been reported. Clinical trials using such peptides are underway now all over the world. Some reports have shown the efficacy of peptide vaccine therapies. However, others ended with unfavorable results, suggesting fundamental underlying problems. One major mechanism that negates the peptide vaccine therapy is tumor escape from immunological systems caused by loss of antigens. TAAs that are used in cancer vaccine therapies may be divided into two major groups: functioning antigens and nonfunctioning antigens. A 'functioning antigen' could be defined as a TAA that is essential for tumor growth, is expressed in several kinds of malignancies and shows homogenous expression in cancerous tissues. It is not difficult to imagine that antigen loss will occur easily with non-functioning antigens as a target of cancer vaccine therapy. Thus, it is essential to use functioning antigens for successful cancer vaccine therapy. In this review, we discuss the functioning antigens and their categorization in detail.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Research on human tumor immunology has greatly advanced in the past two decades. Many immunogenic tumor antigens have been identified, and some of these antigens entered in clinical trials. Consequently, it has been shown that these antigens can inhibit tumor growth in patients to some extent, indicating that they act as potent immunogenic therapeutic vaccines in cancer patients with malignancies originating from various tissues. These patients had antigen-specific cytotoxic T-lymphocyte (CTL) responses when assessed on tetramer, enzyme-linked immunospot (ELISPOT), T-cell clonotype and CTL induction efficiency. Thus, it has become clear that human tumor vaccines can evoke clinical and immunological anti-tumor responses in patients. The tumor regression effects of tumor vaccines, however, are generally low, and it is obvious that current vaccination protocols are generally too weak to provide substantial and satisfactory clinical benefits. This means that other drastic and more potent clinical and immunological protocols are required in cancer immunotherapy. To find such efficient protocols the basic immunological and biological properties of cancers must be investigated. In the present review the identification of human tumor antigens recognized on CTL and the clinical trials are introduced. Next, the most recent analysis of human cancer-initiating cell (cancer stem cell)-associated antigens is described. These antigens might be able to act as 'universal, general and fundamental' tumor antigens. Also present is the authors' recent study for increasing cross-presentation efficiency in dendritic cells and subsequent enhancement of human leukocyte antigen (HLA)-class I-restricted peptide antigenicity by using HSP90 and ORP150 molecular chaperones that act as endogenous Toll-like receptor ligands. In addition to the aforementioned manipulation of the positive loop of tumor immunity, it is necessary to regulate and intervene in the negative loop. In particular, the potential of the expression of HLA class I molecule regulation by epigenetic mechanisms will be discussed. Finally, the type of basic and clinical tumor immunology research highly required currently, and in the very near future, are described.

DOI： 10.1111/j.1440-1827.2009.02353.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cytotoxic T lymphocytes (CTLs) play an essential role in immunologic responses for tumor rejection. In the past decade, various melanoma tumor-associated antigens (TAAs) have been identified, and several clinical trials of vaccination immunotherapy and adoptive immunotherapy using such antigens with or without adjuvants have had fascinating results. However, this has not been the case with oral squamous cell carcinoma (OSCC) because of the difficulty of establishing oral cancer cell lines and CTLs against autologous oral cancer cells. Therefore, few oral cancer antigens have been identified with such CTLs. We herein present the successful establishment of an oral squamous cell carcinoma cell line, POT-1, and an HLA-A24-restricted CTL line (TcPOT-1) from a patient's autologous peripheral blood lymphocytes. TcPOT-1 recognized autologous POT-1 cells in an HLA-A24-restricted manner, and also allogeneic HLA-A24 (+) OSCC cell lines OSC-70 and HSC-2. We also succeeded in isolating two distinct CTL clones from TcPOT-1, HLA-A24-restricted CTL clone 4F11 and HLA-A33-restricted clone 4A11. Both of these clones recognized autologous POT-1 but not allogeneic OSSC cell lines. These data imply that the TcPOT-1 CTL line may include several CTL subpopulations with distinct antigen specificities, such as an HLA-A24-restricted POT-1-specific clone, HLA-A33-restricted POT-1-specific clone, and HLA-A24-restricted allogeneic OSCC-recognizing clone. Therefore, precise analysis of TcPOT-1-recognizing antigens may provide us with important information on as-yet-unknown tumor rejection antigens in OSCC.

DOI： 10.1016/j.humimm.2008.11.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: We previously reported an HLA-A24-restricted cytotoxic T-cell epitope, Survivin-2B80-88, derived from a splice variant of survivin, survivin-2B. In this report, we show a novel HLA-A24-restricted T-cell epitope, Survivin-C58, derived from a wild type survivin, and compared their immunogenicity in oral cancer patients. METHODS: By stimulating peripheral blood lymphocytes of HLA-A24-positive cancer patients with Survivin-C58 peptide in vitro, the peptide-specific CTLs were induced. In order to compare the immunogenic potential between C58 peptide and 2B80-88 peptide, peripheral blood T-cells from thirteen HLA-A24-positive oral cancer patients were stimulated with either or both of these two peptides. RESULTS: Survivin-2B80-88 peptide-specific CTLs were induced from four patients, and C58 peptide-specific CTLs were induced from three out of eight patients with over stage II progression. The CTLs exerted cytotoxicity against HLA-A24-positive tumor cells. In contrast, CTL induction failed from a healthy volunteer and all four patients with cancer stage I. CONCLUSION: It was indicated that a splicing variant-derived peptide and wild type survivin-derived peptide might have a comparable potency of CTL induction, and survivin targeting immunotherapy using survivin-2B80-88 and C58 peptide cocktail should be suitable for HLA-A24+ oral cancer patients.

DOI： 10.1186/1479-5876-7-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: CD8(+) CTLs have an essential role in immune response against tumor. Although tumor-associated antigens have been identified in renal cell carcinoma (RCC), few of these are commonly shared and investigated as therapeutic targets in the clinical medicine. In this report, we show that HIFPH3, a member of prolyl hydroxylases that function as oxygen sensor, is a novel tumor antigen and HIFPH3-specific CTLs are induced from peripheral blood lymphocytes of RCC patients. EXPERIMENTAL DESIGN: Expression of HIFPH3 was examined by reverse transcription-PCR and immunostaining with anti-HIFPH3 antibody. To identify HLA-A24-restricted T-cell epitopes of HIFPH3, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. Peptide-specific CTLs were induced by stimulating peripheral blood lymphocytes of HLA-A24-positive RCC patients with these peptides in vitro. HLA-A24-restricted cytotoxicity of the CTLs against HIFPH3(+) RCC lines was assessed by chromium release assay. RESULTS: HIFPH3 was overexpressed in many RCC cell lines and primary RCC tissues, whereas it was not detectable in normal adult tissues by reverse transcription-PCR. Of the eight peptides that contained HLA-A24-binding motif, HIFPH3-8 peptide (amino acid sequence, RYAMTVWYF) could induce the peptide-specific CTLs from 3 of 6 patients with HIFPH3-positive RCC. Furthermore, HIFPH3-8 peptide-specific CTLs showed cytotoxicity against HIFPH3(+) RCC cell lines in a HLA-A24-restricted manner. CONCLUSIONS: HIFPH3 may be a target antigen in immunotherapy for RCC and HIFPH3-8 peptide could be used as a peptide vaccine for HLA-A*2402(+)/HIFPH3(+) RCC patients.

DOI： 10.1158/1078-0432.CCR-08-0466

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer. METHODS: We set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1-1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks. RESULTS: In the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-gamma responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols. CONCLUSION: This phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

DOI： 10.1186/1479-5876-6-24

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Major histocompatibility complex class I-bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HSC73-binding immunosuppressant, MeDSG disrupted the HSC73-TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Although high-affinity peptide NGT-Bw4 was translocated by TAP, low-affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)-A31-restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I-restricted presentation of HSC73-bound antigenic peptides.

DOI： 10.1111/j.1348-0421.2008.00017.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In rheumatoid arthritis (RA) patients treated with infliximab (IFX), QuantiFERON-TB Gold (QFT-G), an interferon gamma assay for diagnosing tuberculosis infection, was performed to compare its effectiveness to conventional diagnostic procedures (tuberculin skin test, imaging and medical history) in diagnosing latent tuberculosis infection (LTBI). QFT-G was measured bimonthly in 14 rheumatoid arthritis patients during IFX treatment. Seven of 14 patients were confirmed as LTBI positive by at least one method. Of these, four were positive on QFT-G during the study period, and two were positive before the start of IFX administration. For two of the four QFT-G-positive patients, LTBI was diagnosed only by QFT-G. The rate of agreement between QFT-G and conventional procedures was 64.3%. A total of 5% of QFT-G tests were impossible to judge due to decreased reactions in the positive control. These results suggest that QFT-G is able to detect LTBI in RA patients overlooked by conventional methods. Conventional procedures and QFT-G should be employed in parallel, and LTBI should be assumed when one technique gives a positive result.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8(+) T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVES: To assess the expression of livin, a member of the inhibitor of apoptosis protein family, in renal cell carcinoma (RCC) and to determine its prognostic relevance. METHODS: Immunohistochemical staining for livin was performed using paraffin-embedded tissues from 45 cases of RCC. Then we assessed anti-livin antibodies (Abs) in patient sera by enzyme-linked immunosorbent assay and Western blotting. Disease-specific survival of patients was assessed, and differences between the immunohistologically livin-positive and livin-negative groups and between the anti-livin Ab-positive and anti-livin Ab-negative groups were compared with recurrence-free survival using the Kaplan-Meier method and log-rank test. RESULTS: Of the 45 RCC specimens, 26 (57.8%) showed positive staining of livin immunohistochemically. In the RCC patients, anti-livin antibodies were detected and their levels were significantly higher than those in healthy volunteers. However, there was no difference in disease-specific survival between the livin-positive and livin-negative patients or between the anti-livin-positive and anti-livin-negative patients. CONCLUSIONS: Although livin expression may not provide predictive information, it may be recognized as a tumor antigen by the immune system in RCC patients.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DIPA (delta-interacting protein A) was initially identified as a protein that associates with the hepatitis delta antigen. In this study, we found that DIPA can associate with p78/MCRS/MSP58, a Forkhead-associated domain containing protein implicated in malignant transformation as well as in regulation of gene transcription and translation. We analyzed the interaction between DIPA and p78 by co-immunoprecipitation and identified the structural regions involved in the interaction. Consistent with the physical interaction, we found that DIPA is predominant co-localized with p78 to the nucleus. In addition, a fraction of DIPA can be detected on the centrosome. Furthermore, we demonstrate that DIPA can act as a repressor of gene transcription, an activity that appears to be enhanced by p78. Taken together, our results revealed a novel protein complex that plays a role in regulation of gene expression and cell proliferation. We propose that dysfunction of DIPA may contribute to malignant transformation by affecting the functions of p78.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: Various immune systems play important roles in the clinical efficacy of intravesical Bacillus Calmette-Guerin (BCG) instillation for bladder cancer. However, human leukocyte antigen (HLA) class I molecules on tumor cells and various immune system cells infiltrating to/around the tumor have not been evaluated, although many prognostic factors, including clinical, pathologic, and molecular ones, have been investigated. The aim of this study was to determine immunologic prognostic factors of BCG immunotherapy for bladder cancer. EXPERIMENTAL DESIGN: Immunohistochemical staining for HLA class I, CD4, CD8, CD20, CD68, TIA-1, S-100, and FOXP3 was carried out on specimens from 30 patients who underwent BCG immunotherapy from whom both pretreatment and posttreatment specimens were obtained. We did univariate and multivariate analyses of factors affecting recurrence-free survival. The positive, weakly positive, and negative groups of cells that infiltrated to/around the tumor were compared with recurrence-free survival using the Kaplan-Meier method and log-rank test. RESULTS: HLA class I was a significant prognostic factor both in univariate and multivariate analyses. The 5-year recurrence-free survivals of the patients with HLA class I-positive tumors and those with HLA class I-negative tumors were 55.7% and 19.1%, respectively (P = 0.019). There was a significant association between infiltration of CD8, CD20, and CD68-positive cells after BCG therapy and therapeutic effects. CONCLUSIONS: Our data show that expression of HLA class I molecules on tumor cells contributes significantly to the therapeutic effect of BCG immunotherapy for bladder cancer. It is suggested that CTLs may be one of main effectors in this therapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVES: To assess the expression of survivin in transitional cell carcinoma of the bladder and to study whether survivin is a transitional cell carcinoma-specific antigen that could be a target for immunotherapy. Survivin, an inhibitor of apoptosis family member, has been reported to be expressed in various cancers but not in normal adult tissues. METHODS: Immunohistochemical staining for survivin and human leukocyte antigen (HLA) class I was performed on specimens from 88 patients who underwent transurethral resection and radical cystectomy. To determine whether survivin was recognized as a tumor antigen by the host immune system, we assessed anti-survivin antibodies in the sera of 52 patients and 18 healthy volunteers with an enzyme-linked immunosorbent assay using recombinant survivin. RESULTS: Survivin and HLA class I were expressed in 77 (87.5%) and 59 (67.0%) of the 88 bladder cancer specimens, respectively, and 56 (63.6%) expressed both survivin and HLA class I. The absorbance values of anti-survivin antibodies in patients with bladder cancer were significantly greater than those in the healthy volunteers. A relationship was found between the level of serum anti-survivin antibodies and the staining intensity of survivin in the specimen. CONCLUSIONS: Survivin is expressed in carcinoma of the bladder with high sensitivity. It is suggested that survivin is presented on HLA class I molecules of antigen-presenting cells in 64% of patients with bladder cancer. Therapeutic targeting of survivin in bladder cancer is a future possibility.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The centrosome functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation. In our efforts to identify the protein components of the centrosome and to understand the structure features involved in the assembly and functions of this organelle, we cloned and characterized a centrosome-associated protein called Su48. We found that a coiled coil-containing subdomain of Su48 was both sufficient and required for its centrosome localization. In addition, this structure also modulates Su48 dimerization. Moreover, ectopic expression of Su48 causes abnormal mitosis, and a mutant form of Su48 disrupts the localization of gamma-tubulin to the centrosome. Finally, by microinjection of an anti-Su48 antibody, we found that disruption of normal Su48 functions leads to mitotic failure, possibly due to centrosome defects or incomplete cytokinesis. Thus, Su48 represents a previously unrecognized centrosome protein that is essential for cell division. We speculate that Su48 abnormalities may cause aberrant chromosome segregation and may contribute to aneuploidy and malignant transformation.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA-mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16-induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Although human antibody responses to cancer-associated antigens have been detected, the response to livin has not yet been described in lung cancer patients. We examined prevalence of anti-livin antibodies in such patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein. Using a cutoff value for positivity determined as the mean absorbance +2S.D. for healthy control samples, 19 of 37 lung cancer patients (51.3%) were positive for anti-livin antibodies. Of 31 samples from the same lung cancer patients, 18 (58.1%) were positive for anti-survivin antibodies. When sera from 31 lung cancer patients were assessed simultaneously by anti-survivin and anti-livin ELISAs. Twenty-one patients (71%) were positive for survivin, livin, or both. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses. Like anti-survivin antibodies, anti-livin antibodies, thus, can be detected in many lung cancer patients. Testing for both antibodies together may prove useful in detecting lung cancer, but more extensive studies are needed to establish the clinical significance of anti-livin antibodies.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24(+) cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24(+) cancer patients.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

CD8(+) CTLs have an essential role in immune response against tumor. Although an increasing number of tumor-associated antigens that can be recognized by CTLs have been identified from human tumors, a limited number of tumor-associated antigens is known in lung cancer. In addition, because some of them are expressed in noncancerous tissues, there exist limitations in their application to tumor immunotherapy. Livin/ML-IAP is one of recently identified inhibitor of apoptosis protein (IAP) family, which is overexpressed in melanoma cells. In this report, we show that Livin/ML-IAP is aberrantly expressed in many lung cancer cell lines and primary lung cancer tissues, whereas it is not detectable in normal tissues, including lung by reverse transcription-PCR methods. To identify HLA-A24-restricted T-cell epitopes of Livin/ML-IAP, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. It was revealed that Livin7 peptide (amino acid sequence, KWFPSCQFLL) had the highest affinity to HLA-A24. By stimulating peripheral blood lymphocytes of HLA-A24-positive lung cancer patients with Livin7 peptide in vitro, the peptide-specific CTLs were successfully induced from four of five patients with Livin/ML-IAP-positive lung cancer but not from any of four patients without Livin/ML-IAP expression in their cancer tissues. Furthermore, the CTLs induced by Livin7 peptide showed cytotoxicity against Livin/ML-IAP(+) lung cancer cell lines in an HLA-A24-restricted manner. Our data suggest that Livin/ML-IAP may be an excellent target antigen in immunotherapy for lung cancer and Livin7 peptide may serve as a potent peptide vaccine for HLA-A*2402(+)/Livin(+) lung cancer patients.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The prognosis for patients with osteosarcoma who do not respond to current chemotherapy protocols still remains poor. Toward the goal of establishing efficacious peptide-based immunotherapy for those patients, we previously developed an autologous pair of CTLs and an osteosarcoma cell line. In the current study, we screened the cDNA library of this osteosarcoma cell line using an autologous CTL clone and identified cDNA encoding an antigen. The isolated cDNA was identical to papillomavirus binding factor (PBF), which was recently reported as a DNA binding transcription factor cooperating with RUNX1. Reverse transcription-PCR analysis revealed that PBF was expressed in 16 of 19 cases of bone and soft-tissue sarcoma cell lines (5 of 6 of osteosarcoma lines) and 57 of 76 sarcoma tissue samples (11 of 14 of osteosarcoma tissues). Also, PBF was expressed in 10 of 13 epithelial cancer cell lines and 20 of 34 of cancer tissues. In contrast, PBF was detected in some normal organs including ovary, pancreas, spleen, and liver by reverse transcription-PCR but was restricted in the cytoplasm by immunostaining and undetectable by Western blotting. Furthermore, a 12-mer peptide, CTACRWKKACQR, located at the COOH terminus of PBF, was found to be a minimum requirement for recognition by the CTL clone in the context of the HLA-B*5502 molecule. These findings suggest that PBF is a shared tumor-associated antigen, which may serve as a source of peptides applicable to peptide-based immunotherapy for osteosarcoma and other malignant tumors.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Survivin is a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL) recognized by CD8+ cytotoxic T lymphocytes (CTLs). We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2), general malaise (grade 1), and fever (grade 1). No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9) decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer-mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin-induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In this study, we first categorized nine squamous cell carcinoma (SCC) cell lines into two groups in terms of the expression of HLA-DR, -DP, and -DQ molecules. Subsequently, the expression of class II transactivator (CIITA) was studied in these cell lines, because it is widely accepted that the expression of MHC class II molecules is regulated by different types of CIITA transcripts that are initiated by distinct promoters. The majority of the SCC cell lines (six of nine) expressed HLA-DR molecules and CIITA promoter IV (pIV) transcripts in the presence of IFN-gamma. In contrast, three of the nine SCC cell lines were completely negative for class II molecules and all types of CIITA, suggesting epigenetic changes in the promoter region in these cells. Previously, methylation of CIITA pIV was reported to silence CIITA gene expression. We extensively studied the methylation status of CIITA pIV using a panel of 22 SCC cell lines. Remarkably, none of the SCC cell lines demonstrated hypermethylation at the site. In contrast, treatment with a histone deacetylation inhibitor in combination with IFN-gamma clearly restored the expression of the CIITA type IV gene in the HLA-DR-negative SCC cell lines, and the acetylation status of histone H3 examined by chromatin immunoprecipitation analysis was closely associated with the gene expression. Moreover, stable transfection of the CIITA gene into an HLA-DR-negative cell line restored constitutive expression of MHC class II molecules. Therefore, histone deacetylation, but not hypermethylation, modifies CIITA DNA and class II gene expression in SCC.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

INTRODUCTION: We have generated HLA-A*2601-restricted CD8+ CTL clones against an autologous pancreatic cancer cell line. AIMS: To characterize the antigen expressed on the cancer cells. METHODOLOGY: We assessed cytotoxic activities and cytokine production of these CTL clones reacting against cancer cell lines that stably or transiently expressed the HLA-A*2601 gene. RESULTS: These CTL clones recognized 4 of 10 allogeneic pancreatic cancer cell lines and a gallbladder cancer cell line in the context of HLA-A*2601. However, the CTL clones did not recognize three hepatocellular carcinoma cell lines, two esophageal squamous cell carcinoma cell lines, or a lung adenocarcinoma cell line. CONCLUSIONS: Thus, the CTL clones may recognize a shared, but not ubiquitously expressed, tumor antigen on pancreatic and gallbladder cancer cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Because of the difficulty of developing pairs of osteosarcoma cell lines and cytotoxic T lymphocytes (CTLs), no osteosarcoma tumor antigens that are useful for antiosteosarcoma immunotherapy have yet been identified. In parallel with continuous attempts to develop such pairs from osteosarcoma, we employed serological identification using a recombinant expression cloning (SEREX) method to identify B cell-defined antigens. Consequently, a human osteosarcoma cell line, OS2000, was established from a primary osteosarcoma of a patient cured of hereditary retinoblastoma. Repetitious in vitro stimulations by OS2000 cells to the autologous peripheral T cells induced cytotoxic activity in the autologous osteosarcoma cells but not in the nontumor cells. The cytotoxicity was inhibited by anti-HLA class I monoclonal antibody. SEREX analysis revealed that autologous humoral immunity reacted to two proteins expressed in OS2000. One was the self HLA-Cw*0102 molecule, and the other was wild-type smooth muscle myosin light chain (SMMLC). However, no antigenicity of these proteins was seen versus the sera of the other patients. In conclusion, our results demonstrated the presence of host cellular and humoral immune responses to autologous osteosarcoma cells. This offered the opportunity to identify osteosarcoma antigens recognized by autologous immunity.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC-20, was effectively lysed by HLA-DRB1*08032 (HLA-DR8)-restricted autologous CD4+ T cell line, TcOSC-20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC-20 showed a rather strong cytotoxic response to one of the synthetic peptides, namely, amino acid residues 321-336 of human alpha-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recoverin is a photoreceptor-specific calcium binding protein that is only expressed in the retina in normal tissues. Aberrant expression of recoverin, however, has been observed in several cancer tissues and may cause a very rare autoimmune disease, cancer-associated retinopathy (CAR). It has been suggested that CAR-positive cancer patients have a more favorable prognosis than CAR-negative cancer patients. To estimate the status of recoverin-specific T cells in such cancer patients, we generated an HLA-A24-recoverin peptide tetramer. By use of the tetramer, we could directly assess the frequency of CTL precursors (CTLp) of 20 HLA-A24(+) cancer patients with ten colon, six stomach and four breast cancers, and seven healthy individuals. Four cancer patients showed a CTLp frequency higher than 0.9%. Seven cancer patients including the former four patients and two healthy individuals showed specific anti-recoverin cytotoxic responses in an HLA-A24-restricted manner after in vitro stimulation with the recoverin peptide. Moreover, five cancer patients analyzed in an independent experiment using different peripheral blood mononuclear cells (PBMC) samples showed similar CTLp and cytotoxic T lymphocyte (CTL) frequencies and cytotoxic responses, suggesting that the CTLp frequency analyzed by the tetramer and the cytotoxic response may have a good correlation. Thus, we hypothesize that anti-recoverin CTLp may exist in some cancer patients, and that anti-recoverin CTL may be readily induced.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

To date an increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Survivin was originally identified as a member of the inhibitor of apoptosis protein family. Expression of this gene is developmentally regulated. Although survivin is expressed during normal fetal development, the expression is barely detected in terminally differentiated adult tissues except for testis, thymus, and placenta. In contrast, it is abundantly expressed in a wide variety of malignant tissues. We examined the expression of survivin and the two splicing variants survivin-2B and survivin-DeltaEx3 in various cancer cells, immortalized cells, and normal adult tissues. It was demonstrated that two splicing variants were detected in various types of cancer cells as well as survivin, and their expression was more restricted to cancer cells as compared with survivin expression. To identify HLA-A24-restricted T-cell epitopes from survivin and the variant proteins, three peptides were selected from amino acid sequence of these proteins, based on the HLA-A24-binding motif. Peptide binding assay to HLA-A24 revealed that only one peptide designated as survivin-2B80-88 (AYACNTSTL) was capable of binding to HLA-A24. By stimulating peripheral blood lymphocytes with the peptide-pulsed antigen-presenting cells, CTLs were successfully induced in vitro from five of five HLA-A24-positive cancer patients. The CTLs showed significant cytotoxicity against HLA-A24-positive survivin-2B-positive cancer cells. These data suggest that survivin-2B80-88 may be a potent T-cell epitope eliciting CTL response against a splicing variant survivin-2B, which is specifically expressed in many kinds of cancer cells.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously reported acid-extracted natural antigenic peptide (F4.2 [YSWMDISCWI]) of a gastric signet ring cell carcinoma HST-2 cells, recognized by HLA-A*31012-restricted autologous cytotoxic T lymphocytes, TcHST-2 line. In this study, the full-length cDNA (1101 bp), termed c98, predicting a protein composed of 170 amino acids was obtained. Because TcHST-2 cells could lyse the HLA-A31 antigen (+) allogeneic tumor cells that were introduced with c98 gene, this gene was suggested to possess antigenicity. Beginning at N-terminal 61 amino acid, the N-terminal six amino acid sequence that is completely identical to F4.2 was present in c98; however, a sequence of four amino acids in C-terminal was not found. Nevertheless, this peptide, c98(61-70), seemed to be immunogenic, because cells pulsed with c98(61-70) peptide were lysed in a dose-dependent manner by TcHST-2 cells. The c98 gene was expressed ubiquitously in tumor cells as well as in normal tissues. However, some tumor cells, including HST-2 cells, expressed this antigen in a high content, and such cells were lysed by TcHST-2 cells in the context of HLA-A31 antigen. However, TcHST-2 cells did not lyse cells that expressed lower amounts of c98 than HST-2 cells. These data suggested that c98-gene product and/or c98(61-70) peptides could be used as a candidate for tumor vaccines in cancer immunotherapy.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wild-type PTEN protein in the patient's lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient's lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient's lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CD.

PubMed

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