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Immunotherapy based on blockade of the programmed death‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) axis has shown promising clinical activity for renal cell carcinoma (RCC) patients; however, the most effective use of these agents in combination with conventional targeted therapy remains to be resolved. Here we evaluated the therapeutic efficacy of the combination of the mTOR inhibitor everolimus (EVE) and anti‐PD‐L1 using an immunocompetent mouse model of RCC. We first assessed the in vitro effect of EVE on PD‐L1 expression in the human 786‐O and mouse RENCA RCC cell lines and found that EVE upregulated PD‐L1 expression in these RCC cell lines. We then treated RENCA tumor‐bearing mice with EVE and found that PD‐L1 expression was also increased in tumor cells after EVE treatment. To determine the antitumor effects of EVE alone, anti‐PD‐L1 alone, and EVE in combination with anti‐PD‐L1, we evaluated their antitumor effects on RENCA tumor‐bearing mice. A significant decrease in the tumor burden was observed in the EVE alone but not in the anti‐PD‐L1 alone treatment group compared with the control group. Importantly, the combination of EVE with anti‐PD‐L1 significantly reduced tumor burden compared with the EVE alone treatment, increasing tumor infiltrating lymphocytes (TILs) and the ratio of cytotoxic CD8+ T cells to TILs. The results of the present study demonstrated that anti‐PD‐L1 treatment enhanced the antitumor effect of EVE in a mouse model, supporting a direct translation of this combination strategy to the clinic for the treatment of RCC. In this study, we assessed the therapeutic efficacy of the combination of the mTOR inhibitor everolimus (EVE) and anti‐PD‐L1 antibodies using an immunocompetent mouse model of renal cell carcinoma. We also evaluated the correlation between PD‐L1 expression and EVE treatment. Our findings demonstrated that while EVE increased PD‐L1 expression in tumor cells in vivo, a combination of EVE and anti‐PD‐L1 antibody treatment increased tumor‐infiltrating CD8+ T cells and resulted in tumor regression.

Several studies have reported that tissue‐resident memory T cells (TRM cells) or tertiary lymphoid structures (TLSs) are associated with a good prognosis. The aim of this study was to clarify the association of TRM cells and TLSs in the tumor immune microenvironment in gastric cancer (GC). We performed immunohistochemical and immunofluorescence staining to detect the presence of CD103+ T cells and to assess the association between CD103+ T cells and TLSs. CD103+ T cells were observed in the tumor epithelium accompanied by CD8+ T cells and were associated with a better prognosis in GC. Furthermore, CD103+ T cells were located around TLSs, and patients with CD103high had more rich TLSs. Patients who had both CD103high cells and who were TLS‐rich had a better prognosis than patients with CD103low cells and who were TLS‐poor. Moreover, for patients who received PD‐1 blockade therapy, CD103high and TLS‐rich predicted a good response. Flow cytometry was performed to confirm the characteristics of CD103+CD8+ T cells and showed that CD103+CD8+ T cells in GC expressed higher levels of PD‐1, granzyme B, and interferon‐γ than CD103−CD8+ T cells. Our results suggested that CD103+CD8+ cells in GC are correlated with TLSs, resulting in enhanced antitumor immunity in GC. Many CD103+ T cells were observed in the tumor epithelium accompanied by CD8+ T cells and were located around tertiary lymphoid structures (TLSs). Patients with a combination of CD103high and TLS‐rich had a good prognosis.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1 −/− -mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

Background Tertiary lymphoid structures (TLSs) have been reported to be involved in immune responses in many carcinomas. This study investigated the significance of TLSs in esophageal squamous cell carcinoma, focusing on TLS maturation.  Methods The relationships of TLSs with clinicopathological features of 236 patients who underwent curative surgery for stage 0-IV esophageal squamous cell carcinoma were investigated. Mature TLSs, in which the germinal center formation was rich in CD23 + cells, were classified as TLSs containing a germinal center (GC-TLSs). GC-TLS densities were measured, and CD8 + cells were counted. The prognostic impact of GC-TLSs was assessed by Kaplan–Meier plots using the log-rank test for the relapse-free survival. A comparative study of GC-TLSs was performed using the Wilcoxon rank sum test. The relationship between GC-TLSs and CD8 + cells was examined by Spearman’s rank correlation coefficient test. Results TLSs were located mainly at the invasive margin of the tumor in cases with esophageal squamous cell carcinoma. Among the patients treated with neoadjuvant chemotherapy, those with advanced disease had a better prognosis in the GC-TLS high-density group than did those in the GC-TLS low-density group. Patients in whom neoadjuvant chemotherapy was effective had more GC-TLSs than those in whom it was less effective. The density of GC-TLSs and the number of tumor-infiltrating CD8 + cells were higher in patients treated with neoadjuvant chemotherapy than in those without chemotherapy, and a weak correlation between the density of GC-TLSs and the number of tumor-infiltrating CD8 + cells was observed. Moreover, co-culturing of PBMCs with an anticancer drug-treated esophageal squamous cell carcinoma cell line increased the CD20 and CD23 expression in PBMCs in vitro. Conclusion TLS maturation may be important for evaluating the local tumor immune response in patients treated with neoadjuvant chemotherapy for esophageal squamous cell carcinoma. The present results suggest that TLS maturation may be a useful target for predicting the efficacy of immunotherapy, including immune checkpoint inhibitor treatment for esophageal squamous cell carcinoma.

Bioactive peptides from black soldier fly larvae (BSFL) protein hydrolysates have gained attention for their health-promoting properties. Our previous study demonstrated the chemopreventive potential of BSFL hydrolysates prepared with Alcalase (ASBP-AH) in colon cancer cells; their in vivo efficacy has not been fully elucidated. This study evaluated the chemopreventive effects of ASBP-AH, processed by spray-drying (ASBP-AHS) or freeze-drying (ASBP-AHF), in a diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH)-induced rat model of early-stage colorectal carcinogenesis. Oral administration of ASBP-AHS or ASBP-AHF significantly reduced aberrant crypt foci (ACF) and downregulated PCNA, COX-2, and NF-κB expression, without affecting apoptosis. Furthermore, both treatments restored microbial species richness and shifted gut microbial diversity disrupted by carcinogen exposure. ASBP-AHS specifically enriched short-chain fatty acid (SCFA)-producing bacteria, while ASBP-AHF favored anti-inflammatory microbial signatures. Likewise, correlation analysis revealed positive associations between microbial changes and SCFA levels, particularly with ASBP-AHS. Peptidomic profiling identified identical peptides in both hydrolysates, including stable pyroglutamyl-containing sequences with potential anti-inflammatory and microbiota-modulating effects. These findings support the in vivo chemopreventive potential of ASBP-AH and its promise as a functional food ingredient for promoting gut health and reducing colorectal cancer risk.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8–18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

Conjunctival squamous cell carcinoma (SCC) is the most common ocular surface neoplasia. The purpose of this retrospective study was to examine the role of regulatory T cell (Treg) activity in tumor immunity and investigate the tumor microenvironment as a new treatment focus in conjunctival SCC. Cancer progression gene array and immunohistochemical analyses of FOXP3 as a Treg marker, CD8 as a tumor-infiltrating lymphocyte marker, and CXCR4 expression on activated Tregs were conducted in a series of 31 conjunctival SCC cases. The objective was to investigate the immunoreactive response in tumor cells and stromal cells in the cancer microenvironment. The stroma ratio in tumor cells was investigated by monitoring α-smooth muscle actine (SMA) expression between carcinoma in situ (Tis) and advanced carcinoma (Tadv) ( P <0.01). No significant change in PD-L1 expression was observed in this study ( P = 0.15). Staining patterns of FOXP3, CD8, and CXCR4 were examined separately between tumor cells and stromal cells in SCC tumors. Differences in staining of FOXP3 in Tregs and CD8 in tumor-infiltrating lymphocytes in tumor stroma in the Tis group were observed compared with the Tadv group (each P <0.01). In addition, double immunostaining of CXCR4/FOXP3 was correlated with progression-free survival ( P = 0.049). Double immunostaining of CXCR4/FOXP3 correlated with American Joint Committee on Cancer T-stage, independent of age or Ki67 index ( P <0.01). Our results show that FOXP3 and the CXCR4/FOXP3 axis are important pathologic and prognostic factors of ocular surface neoplasia, including SCC. The tumor microenvironment of conjunctival SCC should be considered in the future development of treatment options.

BackgroundRadiotherapy (RT) has recently been highlighted as a partner of immune checkpoint inhibitors. The advantages of RT include activation of lymphocytes while it potentially recruits immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). This study aimed to investigate the mechanism of overcoming treatment resistance in immunologically cold tumours by combining RT and MDSC-targeted therapy.MethodsThe abscopal effects of irradiation were evaluated using MB49 and cisplatin-resistant MB49R mouse bladder cancer cells, with a focus on the frequency of immune cells and programmed cell death-ligand 1 (PD-L1) expression in a xenograft model.ResultsMB49R was immunologically cold compared to parental MB49 as indicated by the fewer CD8+ T cells and lower PD-L1 expression. Polymorphonuclear MDSCs increased in both MB49 and MB49R abscopal tumours, whereas the infiltration of CD8+ T cells increased only in MB49 but not in MB49R tumours. Interestingly, PD-L1 expression was not elevated in abscopal tumours. Finally, blocking MDSC in combination with RT remarkably reduced the growth of both MB49 and MB49R abscopal tumours regardless of the changes in the frequency of infiltrating CD8+ T cells.ConclusionsThe combination of RT and MDSC-targeted therapy could overcome treatment resistance in immunologically cold tumours.

Objectives: Our aim was to report integrative evidence for the health risk assessment of ortho-toluidine (OT) in bladder cancer in a mini-review of the recent studies of humans, experimental animals, and OT skin permeability.Methods: Bladder cancer cases were identified in workers in Japan who were occupationally exposed in 2014-2017 to aromatic amines, primarily to the human carcinogen OT.Results: A key epidemiological study of 98 aromatic amine-exposed workers in Japan showed a clear OT exposure-response relationship with a standardized incidence ratio. A rat model experimental study also indicated that OT and acetoaceto-o-toluidine are potent bladder carcinogens. Multiple mechanisms of OT-related bladder cancer have been proposed: metabolic activation to reactive metabolites that bind DNA and proteins, mutagenicity, oxidative DNA damage, chromosomal damage, and cytotoxicity by OT. Recent comprehensive analyses of DNA adducts in rats identified a number of common oxidative DNA adducts, including 8-OHdG, in the rat urothelium and indicated that oxidative stress may play a crucial role in the development of urinary cancer caused by OT. The skin permeability of 6 aromatic amines (o-toluidine, aniline, p-toluidine, o-anisidine, 2,4-dimethylaniline, and o-chloroaniline) was examined with the use of a 3-dimensional (3D) reconstructed human skin model; ~70%-80% of the 6 aromatic amines had permeated through the 3D skin within 8 hours. Genotoxic potency testing in a human urothelial cell line using γ-H2AX, a marker of DNA damage, suggested that OT exhibited strong γ-H2AX generation.Conclusions: Prolonged dermal exposure to OT along with other genotoxic aromatic amines over many years may contribute to the development of bladder cancer.

To uncover mechanisms and explore novel biomarkers of obesity, type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH)-associated hepatocarcinogenesis, cellular and molecular alterations in the liver, and hepatocellular carcinomas (HCCs) were investigated in NASH model 60-week-old Tsumura, Suzuki, Obese Diabetic (TSOD) mice and NASH HCC patients. Markedly elevated lipid deposition, inflammation, fibrosis, and peroxisome proliferation in the liver, preneoplastic lesions, and HCCs of TSOD mice were accompanied by accumulation of polysaccharides in the cellular cytoplasm and nuclei and increase of oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG) formation in the liver and altered foci. Metabolomics of TSOD mice HCCs demonstrated significant elevation of the concentration of amino acid L-arginine, phosphocreatine, S-adenosylmethionine/S-adenosylhomocysteine ratio, adenylate, and guanylate energy charges in coordination with tremendous rise of glucose metabolites, mostly fructose 1,6-diphosphate. L-arginine accumulation in HCCs was associated with significant under-expression of arginase 1 (ARG1), suppression of the urea cycle, methionine and putrescine degradation pathways, activation of Ser and Thr kinase Akt AKT, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, β-catenin, mammalian target of rapamycin (mTOR), and cell proliferation. Furthermore, clinicopathological analysis in 20 metabolic syndrome/NASH and 80 HCV-positive HCC patients demonstrated significant correlation of negative ARG1 expression with poor tumor differentiation, higher pathological stage, and significant decrease of survival in metabolic syndrome/NASH-associated HCC patients, thus indicating that ARG1 could become a potential marker for NASH HCC. From these results, formation of oxidative stress and 8-OHdG in the DNA and elevation of glucose metabolites and L-arginine due to ARG1 suppression in mice liver cells are the important characteristics of T2DM/NASH-associated hepatocarcinogenesis, which may take part in activating oxidative stress resistance, synthesis of phosphocreatine, cell signaling, methylation, and proliferation.