Restoration of ARID1A Protein in ARID1A-deficient Clear Cell Carcinoma of the Ovary Attenuates Reactivity to Cytotoxic T Lymphocytes.

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.

Assessment of cancer cell-expressed HLA class I molecules and their immunopathological implications.

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.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

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.

CD8-positive T Cell Infiltration With Human Leukocyte Antigen Class 1 Expression Predicts Patients With Stage IV Colorectal Cancer Survival.

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.

Prognostic significance of immunohistochemical classification utilizing biopsy specimens in patients with extensive-disease small-cell lung cancer treated with first-line chemotherapy and immune checkpoint inhibitors.

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.

NF9 peptide specific cytotoxic T lymphocyte clone cross react to Y453F mutation of SARS-CoV-2 virus spike protein.

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.

Development of T cell receptor-engineered T cells targeting the sarcoma-associated antigen papillomavirus binding factor.

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 ß 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.

Eribulin is an immune potentiator in breast cancer that upregulates human leukocyte antigen class I expression via the induction of NOD-like receptor family CARD domain-containing 5.

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.

Establishment and characterization of a novel dedifferentiated chondrosarcoma cell line, SMU-DDCS, harboring an IDH1 mutation.

Dedifferentiated chondrosarcoma (DDCS) is a high-grade subtype with a bi-morphic histological appearance of a conventional chondrosarcoma component and it can abruptly transition to a high-grade non-cartilaginous sarcoma. To better understand the biological features of DDCSs and to help develop new therapies, a novel DDCS cell line, SMU-DDCS, was established. Tissue from an open biopsy of a tumor resected from a 75-year-old patient was subjected to primary culture. The cell line was established and authenticated by assessing DNA microsatellite short tandem repeats. The cells maintained in monolayer cultures exhibited constant growth, spheroid formation, and high invasive capacity. Out of the four mice inoculated with SMU-DDCS cells, tumors developed in three mice after 2 weeks. R132C mutation was found in the IDH1 but not the IDH2 genomic DNA sequence of SMU-DDCS cells. SMU-DDCS cells exhibited low chemosensitivity to doxorubicin, methotrexate, and cisplatin. This SMU-DDCS cell line harboring an IDH1 mutation will be a useful tool for investigating DDCS development and for evaluating novel therapeutic agents against it.

Exploring olfactory receptor family 7 subfamily C member 1 as a novel oral cancer stem cell target for immunotherapy.

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.

Fusion with type 2 macrophages induces melanoma cell heterogeneity that potentiates immunological escape from cytotoxic T lymphocytes.

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.

SOX10 Inhibits T Cell Recognition by Inducing Expression of the Immune Checkpoint Molecule PD-L1 in A375 Melanoma Cells.

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.

Metastatic pulmonary pleomorphic carcinoma replaced by a granulomatous lesion after spontaneous regression and PD-1 blockade-induced regression: can epithelioid granuloma be a histological hallmark of cancer immunity?

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.

Cisplatin resistance driver claspin is a target for immunotherapy in urothelial carcinoma.

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.

Immunohistochemical analysis of arachidonate 5-lipoxygenase expression in B-cell lymphomas: Implication for B cell differentiation and its analogy with lymphomagenesis.

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.

Development of CAR-T cells specifically targeting cancer stem cell antigen DNAJB8 against solid tumours.

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.

Cisplatin-induced HSF1-HSP90 axis enhances the expression of functional PD-L1 in oral squamous cell carcinoma.

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.

Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry.

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.

High aldehyde dehydrogenase 1 activity is related to radiation resistance due to activation of AKT signaling after insulin stimulation in prostate cancer.

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.

Tumor-infiltrating CD8+ T cells recognize a heterogeneously expressed functional neoantigen in clear cell renal cell carcinoma.

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.