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Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8+ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs. We found that, TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. And, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio. Inhibition of TGF‐β restores the immunosuppressive TME to active status by a mechanism different from that of immune‐checkpoint inhibitors, suggesting that the combination of both may lead to a new therapeutic strategy for OSCCs.

Background Upward-directed exit-site has been believed to be the worst for frequent ESI by an old retrospective study using straight catheters. No comparison study of 3 exit-site directions using swan-neck catheter has been performed regarding which direction is the best for our endpoints, Easy-to-see the backside area of exit-site: ESBE, Easy-to-disinfect the backside area of exit-site: EDBE, reduction of both exit-site infection (ESI), symptomatic catheter dislocation and peritonitis. Methods We assessed the relationship of exit-site direction with our endpoints in a quantitative cross-sectional, multicentered questionnaire survey. Patients who received either non-surgical catheter implantation or exit-site surgery were excluded. Results The numbers (percentage) of exit-site directions in included 291 patients were upward 79 (26.0), lateralward 108 (37.5) and downward 105 (36.5). Cochran-Armitage analysis showed a significant step-ladder increase in the prevalence of ESI as the direction changed from upward to lateralward to downward (0.15 ± 0.41, 0.25 ± 0.54, 0.38 ± 0.69 episodes/patient-year, p  = 0.03). Multivariable regression analysis revealed the upward exit-site independently associates with both higher frequency of ESBE (OR 5.55, 95% CI 2.23–16.45, p  < 0.01) and reduction of prevalence of ESI (OR 0.55, 95%CI 0.27–0.98, p  = 0.04). Positive association between the prevalence of symptomatic catheter dislocation and ESI (OR 2.84, 95% CI 1.27–7.82, p  = 0.01), and inverse association between EDBE and either prevalence of symptomatic catheter dislocation (OR 0.27, 95% CI 0.11–0.72) or peritonitis (OR 0.48, 95% CI 0.23–0.99) observed. Conclusion Upward-directed swan-neck catheter exit-site may be the best for both ESBE and prevention of ESI. EDBE may reduce catheter dislocation and peritonitis. Symptomatic catheter dislocation may predict ESI.

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1+ T‐cells interacted with PD‐L1+ cancer cells or PD‐L1+ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC. Frequencies of eTregs populations (CD45RA‐FOXP3hi) in CD4+ T‐cells of PBL and TIL from HNSCC patients were compared. High frequency of eTreg infiltration into HNSCC tissues was revealed. In addition, MFI of CD25 and FOXP3 in eTreg of TIL were higher than in PBL, which indicates that eTreg in TIL are not only highly infiltrated into the tissues but also are highly activated.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia‐responsive tumor‐associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9‐expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a “cold” tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen‐specific cytotoxic T cells compared with RENCA cells. Notably, siRNA‐mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA‐mediated knockdown of the mouse CA9 gene inhibited the hypoxia‐induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia‐associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as “cold.”

Docetaxel (DOC) is one of the most effective chemotherapeutic agents against castration-resistant prostate cancer (CRPC). Despite an impressive initial clinical response, the majority of patients eventually develop resistance to DOC. In tumor metabolism, where tumors preferentially utilize anaerobic metabolism, lactate dehydrogenase (LDH) serves an important role. LDH controls the conversion of pyruvate to lactate, with LDH-A, one of the predominant isoforms of LDH, controlling this metabolic process. In the present study, the role of LDH-A in drug resistance of human prostate cancer (PC) was examined by analyzing 4 PC cell lines, including castration-providing strains PC3, DU145, LNCaP and LN-CSS (which is a hormone refractory cell line established from LNCaP). Sodium oxamate (SO) was used as a specific LDH-A inhibitor. Changes in the expression level of LDH-A were analyzed by western blotting. Cell growth and survival were evaluated with a WST-1 assay. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V staining. LDH expression was strongly associated with DOC sensitivity in PC cells. SO inhibited growth of PC cells, which was considered to be caused by the inhibition of LDH-A expression. Synergistic cytotoxicity was observed by combining DOC and SO in LN-CSS cells, but not in LNCaP cells. This combination treatment induced additive cytotoxic effects in PC-3 and DU145 cells, caused cell cycle arrest in G2-M phase and increased the number of cells in the sub-G1 phase of cell cycle in LN-CSS cells. SO promoted DOC induced apoptosis in LN-CSS cells, which was partially caused by the inhibition of DOC-induced increase in LDH-A expression. The results strongly indicated that LDH-A serves an important role in DOC resistance in advanced PC cells and inhibition of LDH-A expression promotes susceptibility to DOC, particularly in CRPC cells. The present study may provide valuable information for developing targeted therapies for CRPC in the future.

Regulatory T-cells (Tregs) play a major role in suppressing anti-tumor immune responses. Mogamulizumab, an anti-CC chemokine receptor type 4 (CCR4) monoclonal antibody, depletes effector Tregs (eTregs). However, the clinical efficacy of mogamulizumab was limited in phase Ia/Ib studies for solid tumors (NCT01929486); the finding suggests that mogamulizumab may also deplete beneficial CCR4 + CD8 + T-cells in patients. Therefore, we focused on CTLs and aimed to identify a way to protect CCR4 + CTLs. Here, we evaluated the association of CCR4 expression in cytotoxic T-lymphocytes (CTLs) with antigen and cytokine stimulations and kinase inhibition using cytomegalovirus antigen instead of tumor antigen. CCR4 expression in CTLs was induced by antigen stimulation (mean 3.14–29.0%), enhanced by transforming growth factor-β1 (TGF-β1) (mean 29.0–51.2%), and downregulated by trametinib with (mean 51.2–11.4%) or without TGF-β1 treatment (mean 29.0–6.98%). Phosphorylation of ERK in CD8 + T-cells was suppressed by trametinib. Regarding the effect on immunological function of CTL, trametinib reduced cytokine production but not affected cytotoxicity. Importantly, trametinib alleviated CTL reduction by anti-CCR4 antibody without affecting eTreg depletion because CCR4 expression in eTregs was not downregulated. In conclusion, combination therapy with trametinib may improve the clinical efficacy of mogamulizumab.

In this paper, we show that every antisymmetric multiple stochastic (Ito’s) integral has a polynomial form of single and double ones. As an application, a new approximating scheme for the solution to a stochastic differential equation is proposed.

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Objectives Squamous cell carcinoma is the most common malignancy in the oral cavity. Moreover, human papillomavirus (HPV) infection has been recently implicated in the onset of oral squamous cell carcinoma (OSCC). Regulatory T cells (Tregs) are Forkhead box P3 (FoxP3) positive and are normally involved in the mechanism by which organisms escape attacks from their own immune system; however, in tumors, these cells are known to suppress antitumor immunity and block the attack against tumors. The present study evaluated the associations of the number of Tregs and HPV infection with prognoses in patients with OSCC. Material and methods Samples from 106 patients diagnosed with OSCC were evaluated by immunohistochemical staining for the identification of FoxP3+ Tregs and HPV. The relationship between the observed number of Foxp3‐positive cells, the presence/absence of HPV infection and associations with clinicopathological indicators were analyzed. Results Tissues were classified into high (High) and low (Low) Treg count groups, with 69 patients classified as High and 37 classified as Low. The prognoses were significantly better in the Low group compared with the High group (p = 0.04). FoxP3 expression may have had some effect on nodal metastases (p = 0.09). HPV antigens were detected in 65 patients, but there were no significant associations with prognosis (p = 0.34). HPV‐infected tumors were more common in the gums and tongues than in the lips, cheeks, and floor of the mouth (p = 0.05). Conclusions These results indicate that Tregs in tumor sites are associated with worsened prognoses of patients with OSCC and suggest potential therapies targeting Tregs in OSCC.

Patients with advanced/recurrent mucoepidermoid carcinoma (MEC) have a poor prognosis. This study aimed to establish and characterize human mucoepidermoid carcinoma cell lines from the initial surgical specimen and biopsy specimen upon recurrence from the same patient to provide a resource for MEC research. MEC specimens from the initial surgical procedure and biopsy upon recurrence were used to establish cell lines. The established cell lines were cytogenetically characterized using multi-color fluorescence in situ hybridization and detection, and the sequence of the CRTC1-MAML2 chimeric gene was determined. Furthermore, the susceptibility of head and neck mucoepidermoid carcinoma to standard treatment drugs such as cisplatin, 5-fluorouracil, and cetuximab was investigated. We successfully established unique MEC cell lines, AMU-MEC1, from an initial surgical specimen and AMU-MEC1-R1 and AMU-MEC1-R2 from the recurrent biopsy specimen in the same patient. These cell lines exhibited epithelial morphology and developed in vitro-like cobblestones. They shared eight chromosomal abnormalities, including der(19)ins(19;11)(p13;?), which resulted in a chimeric CRTC1-MAML2 gene, indicating the same origin of the cell lines. The susceptibility of all cell lines to cisplatin and 5-fluorouracil was low. Interestingly, EGFR dependency for cell growth decreased in AMU-MEC-R1 and AMU-MEC-R2 but was retained in AMU-MEC1. These cytogenetic and biochemical findings suggest that the established cell lines can be used to investigate the disease progression mechanisms and develop novel therapeutics for MEC.