Regulatory T-cells activated in metastatic draining lymph nodes possibly suppress cancer immunity in cancer tissues of head and neck squamous cell cancer.

Regulatory T cells (Tregs) play an important role in creating an immunosuppressive microenvironment in cancer tissues. However, the mechanisms by which Tregs are activated and suppress cancer immunity remain unclear. To elucidate these mechanisms, we performed a T cell receptor (TCR) repertoire analysis of Tregs and conventional T cells in peripheral blood, draining lymph nodes (DLNs), and cancer tissues of patients with head and neck squamous cell cancer (HNSCC). We found that the TCR repertoire was skewed in cancer tissue and metastatic DLNs (M-DLNs) compared with non-metastatic DLNs, and TCR repertoire similarities in Tregs and CD8+ T cells between M-DLNs and cancer tissue were high compared with those at other sites. These results suggest that Tregs and CD8+ T cells are activated in M-DLNs and cancer tissues by cancer antigens, such as neoantigens, and shared antigens and Tregs suppress CD8+ T cell function in a cancer antigen-specific manner in M-DLNs and cancer tissue. Moreover, M-DLNs might be a source of Tregs and CD8+ T cells recruited into the cancer tissue. Therefore, targeting Tregs in M-DLNs in an antigen-specific manner is expected to be a novel immunotherapeutic strategy for HNSCCs.

Upward-directed exit-site of the swan-neck catheter and "Easy-to-disinfect the backside area of exit-site" may prevent PD complications.

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.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

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."

A multi-institutional, observational study of outcomes after catheter placement for peritoneal dialysis in Japan.

BACKGROUND: This multi-institutional, observational study examined whether the outcomes after peritoneal dialysis (PD) catheter placement in Japan meet the audit criteria of the International Society for Peritoneal Dialysis (ISPD) guideline and identified factors affecting technique survival and perioperative complications. METHODS: Adult patients who underwent first PD catheter placement for end-stage kidney disease between April 2019 and March 2021 were followed until PD withdrawal, kidney transplantation, transfer to other facilities, death, 1 year after PD start or March 2022, whichever came first. Primary outcomes were time to catheter patency failure and technique failure, and perioperative infectious complications within 30 days of catheter placement. Secondary outcomes were perioperative complications. Appropriate statistical analyses were performed to identify factors associated with the outcomes of interest. RESULTS: Of the total 409 patients, 8 who underwent the embedded catheter technique did not have externalised catheters. Of the 401 remaining patients, catheter patency failure occurred in 25 (6.2%). Technical failure at 12 months after PD catheter placement calculated from cumulative incidence function was 15.3%. On Cox proportional hazards model analysis, serum albumin (hazard ratio (HR) 0.44; 95% confidence interval (CI) 0.27-0.70) and straight type catheter (HR 2.14; 95% CI 1.24-3.69) were the independent risk factors for technique failure. On logistic regression analysis, diabetes mellitus was the only independent risk factor for perioperative infectious complications (odds ratio 2.70, 95% CI 1.30-5.58). The occurrence rate of perioperative complications generally met the audit criteria of the ISPD guidelines. CONCLUSION: PD catheter placement in Japan was proven to be safe and appropriate.

Artesunate and cisplatin synergistically inhibit HNSCC cell growth and promote apoptosis with artesunate­induced decreases in Rb and phosphorylated Rb levels.

In the treatment of head and neck cancer, cisplatin is often used as a therapeutic agent; however, its efficacy is limited and it can cause renal dysfunction as an adverse effect. For this reason, the use of cisplatin is limited in elderly patients with reduced renal function. Recently, artemisinin, which was developed as an antimalarial drug, was found to have antitumor effects and is effective in combination with other anticancer drugs. In the present study, the antitumor effects of artemisinin and its derivatives as well as their combination with cisplatin and iron on head and neck squamous cell carcinoma cell lines, were investigated. Cell viability was determined by a cell viability assay, the cell cycle was analyzed by flow cytometry, cell death was assessed with annexin V and propidium iodide staining, and western blotting was used to analyze retinoblastoma protein (Rb), phosphorylated (p­)Rb, and other cell cycle­associated molecules. A total of four artemisinin compounds were examined and it was found that artesunate and dihydroartemisinin had a significant inhibitory effect on growth. It was also identified that the combination of artesunate, cisplatin, and iron inhibited cell proliferation and caused S/G2­M cell cycle arrest. In addition, western blotting of Rb, a molecule involved in the cell cycle, showed that artesunate induced the loss of not only Rb but also p­Rb. These results suggested that artesunate is a useful drug in combination with cisplatin.

Pilot study of plasma creatine riboside as a potential biomarker for cervical cancer.

This pilot study aimed primarily to evaluate plasma levels of a novel metabolite, creatine riboside, in patients with cervical cancer (discovery and validation cohorts, n = 11 for each) compared with non-cancer subjects (controls, n = 30). We found that the pre-treatment plasma creatine riboside level was significantly higher in the discovery cohort than in controls. The cut-off value determined from the discovery cohort distinguished 90.9% of the patients in the validation cohort from controls. Unbiased principal component analysis of plasma metabolites in high-creatine riboside samples demonstrated enrichment of pathways involved in arginine and creatine metabolism. These data indicate the potential utility of plasma creatine riboside as a biomarker of cervical cancer.

Moyamoya disease-specific extracellular vesicle-derived microRNAs in the cerebrospinal fluid revealed by comprehensive expression analysis through microRNA sequencing.

PURPOSE: To examine the specific changes that occur in the expression levels of extracellular vesicle-derived microRNAs (miRNAs) in intracranial cerebrospinal fluid (CSF) in moyamoya disease. METHODS: Patients with arteriosclerotic cerebral ischemia were used as controls to eliminate the effects of cerebral ischemia. Intracranial CSF was collected from moyamoya disease and control patients during bypass surgery. Extracellular vesicles (EVs) were extracted from the CSF. Comprehensive expression analysis of miRNAs extracted from EVs by next-generation sequencing (NGS) and validation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed. RESULTS: Experiments were conducted on eight cases of moyamoya disease and four control cases. In the comprehensive miRNA expression analysis, 153 miRNAs were upregulated, and 98 miRNAs were downregulated in moyamoya disease compared to the control cases (q-value < 0.05 and |log2 fold change|> 1). qRT-PCR performed on the four most variable miRNAs (hsa-miR-421, hsa-miR-361-5p, hsa-miR-320a, and hsa-miR-29b-3p) associated with vascular lesions among the differentially expressed miRNAs gave the same results as miRNA sequencing. On gene ontology (GO) analysis for the target genes, cytoplasmic stress granule was the most significant GO term. CONCLUSIONS: This study is the first comprehensive expression analysis of EV-derived miRNAs in the CSF of moyamoya disease patients using NGS. The miRNAs identified here may be related to the etiology and pathophysiology of moyamoya disease.

Renal injury associated with MYH9 disorder with 5773delG mutation: A case report.

A 35-year-old man with persistent urine abnormalities and renal dysfunction was referred to our hospital. May-Hegglin anomaly was suspected, and a renal biopsy showed focal segmental glomerulosclerosis (FSGS) with IgA deposition. Electron microscopy revealed foot process effacements and intense bleb-like morphological changes in podocytes. Nonmuscle myosin heavy chain IIA (NMMHCIIA) staining of granulocytes revealed a localized, type II pattern, and genomic DNA sequencing of MYH9 exon 40 revealed MYH9 5773delG mutation (c.5773delG [p.(Asp1925Thrfs*23)]). Podocytes were significantly stained by an antibody specific for NMMHC-IIA abnormalities associated with this mutation. Colocalization observation of vimentin and NMMHC-IIA demonstrated a diminished form of NMMHC-IIA in podocytes. Taking these observations into account, it was determined that the present case was likely associated with MYH9 disorder. Treatment was started with olmesartan, followed by methylprednisolone pulse therapy 3 times bi-monthly. Finally, the patient began hemodialysis 18 months later. This is the first known report of renal phenotype expression associated with this MYH9 mutation. FSGS can occur in association with MYH9 mutations at the 3' regions, such as exon 40. Abnormal expression or metabolism of NMMHC-IIA in podocytes might be related to the formation of FSGS lesions due to this MYH9 mutation.

Establishment of Mucoepidermoid Carcinoma Cell Lines from Surgical and Recurrence Biopsy Specimens.

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.

Long noncoding RNA profile of the intracranial artery in patients with moyamoya disease.

OBJECTIVE: Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive stenosis of the internal carotid artery (ICA) and secondary formation of collateral vessels. Revascularization surgery is performed in patients with MMD to prevent stroke; however, the pathogenesis of MMD remains unknown. Recently, long noncoding RNAs (lncRNAs) have been found to play a key role in gene regulation and are implicated in various vascular diseases. However, the lncRNA expression profile in MMD lesions has not been investigated. In this study the authors aimed to determine the characteristics of lncRNA expression in MMD lesions. METHODS: The authors collected microsamples of the middle cerebral artery (MCA) from patients with MMD (n = 21) and patients with control conditions (n = 11) who underwent neurosurgical treatment. Using microarray experiments, the authors compared the profiles of lncRNA expression in the MCAs of the MMD and control patient groups and identified differentially expressed lncRNAs (fold change > 2, q < 0.05). In addition, the neighboring coding genes, whose transcription can be regulated in cis by the identified differentially expressed lncRNAs, were investigated and Gene Ontology (GO) analysis was applied to predict associated biological functions. RESULTS: The authors detected 308 differentially expressed lncRNAs (fold change > 2, q < 0.05), including 306 upregulated and 2 downregulated lncRNAs in the MCA from patients with MMD. Regarding the prediction of biological function, GO analyses with possible coding genes whose transcription was regulated in cis by the identified differentially expressed lncRNAs suggested involvement in the antibacterial humoral response, T-cell receptor signaling pathway, positive regulation of cytokine production, and branching involved in blood vessel morphogenesis. CONCLUSIONS: The profile of lncRNA expression in MMD lesions was different from that in the normal cerebral artery, and differentially expressed lncRNAs were identified. This study provides new insights into the pathophysiology of MMD.

Trametinib improves Treg selectivity of anti-CCR4 antibody by regulating CCR4 expression in CTLs in oral squamous cell carcinoma.

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-ß1 (TGF-ß1) (mean 29.0-51.2%), and downregulated by trametinib with (mean 51.2-11.4%) or without TGF-ß1 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.

Renal thrombotic microangiopathy and nephrotic proteinuria induced by intravitreal injection of aflibercept for diabetic macular edema.

BACKGROUND: Vascular endothelial growth factor inhibitors (VEGFIs) are used to treat malignant neoplasms and ocular diseases by inhibiting angiogenesis. Systemic use of VEGFIs has various side effects, including hypertension, proteinuria, and thrombotic microangiopathy, but adverse events due to intravitreal injection of VEGFIs have not been fully clarified. Although age-related macular degeneration was initially the most common target of intravitreal injection of VEGFIs, it has also been applied sporadically for diabetic macular edema in recent years. Proteinuria following intravitreal injection of VEGFIs would be reversible. In patients with diabetes mellitus (DM), however, it would be difficult to determine whether kidney damage arises from the clinical course of DM or from intravitreal injection of VEGFIs for diabetic macular edema. CASE PRESENTATION: A 55-year-old woman with a 20-year history of type 2 DM began intravitreal injection of VEGFI (aflibercept, 2 mg every 4 weeks) for treatment of diabetic macular edema 2 years previously. She presented with leg edema, hypertension, and nephrotic-range proteinuria 14 months after the first injection. Histological examination of renal biopsy specimens revealed diabetic nephropathy with renal thrombotic microangiopathy probably associated with intravitreal injection of VEGFI. The patient's nephrotic syndrome completely improved at 6 months after simply discontinuing aflibercept. CONCLUSIONS: This is a precious report of pathologically investigated renal thrombotic microangiopathy leading to nephrotic syndrome due to intravitreal injection of aflibercept for diabetic macular edema in a patient with type 2 DM. Renal function and proteinuria should be monitored in diabetic patients who receive intravitreal injection of a VEGFI. If kidney damage develops independent of the clinical course of DM during intravitreal injection of a VEGFI, renal biopsy should be performed and intravitreal VEGFI injection discontinued.

Interleukin-6 blockade reduces salt-induced cardiac inflammation and fibrosis in subtotal nephrectomized mice.

Cardiovascular disease is the most common comorbidity in patients with chronic kidney disease (CKD), affecting both their prognosis and quality of life. Cardiac fibrosis is common in patients with CKD with left ventricular diastolic dysfunction, and it is associated with increased risk of heart failure and mortality. Recent evidence suggests that high salt intake activates immune responses associated with local accumulation of sodium. We reported that high salt intake promotes cardiac inflammation in subtotal nephrectomized (Nx) mice. We investigated the effects of administration of MR16-1, a rat anti-mouse monoclonal interleukin (IL)-6 receptor antibody, in Nx mice with salt loading (Nx-salt). Expression of monocyte chemoattractant protein-1, tumor necrosis factor-α, IL-1ß, and IL-6 mRNAs and macrophage infiltration was significantly reduced in the heart of Nx-salt mice treated with MR16-1 (Nx-salt-MR16-1) compared with Nx-salt mice treated with control rat rat IgG1 (Nx-salt-rat IgG1). Correspondingly, cardiac fibrosis was significantly attenuated in Nx-salt-MR16-1 mice compared with Nx-salt-rat IgG1 mice. Furthermore, in the heart of Nx-salt-MR16-1 mice, expression of mRNA for nicotinamide adenine dinucleotide phosphate oxidase-2, an oxidative stress marker, was significantly downregulated compared with Nx-salt-rat IgG1 mice. Increases in cardiac metabolites, including histidine and γ-butyrobetaine, were also reversed by IL-6 blockade treatment. In conclusion, IL-6 blockade exerts anti-inflammatory, antifibrotic, and partial antioxidative effects in the heart of Nx-salt mice.NEW & NOTEWORTHY In the present study, IL-6 blockade exerted anti-inflammatory, antifibrotic, and partial antioxidative effects on the hearts of mice with CKD on a high-salt diet. Therefore, IL-6 potentially mediates cardiac fibrosis induced by high salt intake in patients with CKD, a finding with therapeutic implications. Of note, the next therapeutic implication may simply be the reinforcement of low-salt diets or diuretics and further research on the anti-inflammatory effects of these measures rather than IL-6 blockade with high-salt diet.

An In Vivo Study of Local Administration of Low-dose Anti-PD-1 Antibody Using an Oral Cancer Cell Line.

BACKGROUND/AIM: The immunotherapy approach using anti-programmed cell death 1 (PD-1) antibody has been demonstrated in oral cancer treatment. However, serious immune-related adverse events (irAE) have been reported. If local administration of small doses of anti-PD-1 antibody via intraarterial chemoradiotherapy could have the same antitumor effect of systemic administration, this can reduce irAE and medical expenses. In this study, we investigate the antitumor effects of local and systemic administration of a small amount of anti-PD-1 antibody, the overall survival (OS), and the immune environment around cancer. MATERIALS AND METHODS: A mouse buccal mucosal oral squamous cell carcinoma cell line (Sq-1979) was used, and anti-mouse PD-1 was used as the drug. The cell line was transplanted to mouse, and the drug was locally (30 mg/body) and systemically (300 mg/body) administered in a dose. The tumor shrinkage rate and antitumor effect were examined 21 and 29 days after the start of administration. The OS was also compared in each of the groups. Furthermore, the subcutaneous growth of the tumors was inhibited, and the expressions of PD-L1, CD8T cells, perforin, and granzyme B were examined. RESULTS: We found that the local low-dose and systemic groups had the same antitumor effect, OS also showed a significant prolongation. In addition, indicated that the expression of granzyme B was higher in the local low-dose group. CONCLUSION: Local low-dose administration of anti-PD-1 antibody showed the same antitumor effect and OS as systemic normal-dose administration. Therefore, local low-dose administration in oral cancer can be useful.

In Situ PD-L1 Expression in Oral Squamous Cell Carcinoma Is Induced by Heterogeneous Mechanisms among Patients.

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.

Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma.

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.

T-cell responses and combined immunotherapy against human carbonic anhydrase 9-expressing mouse renal cell carcinoma.

Renal cell carcinoma (RCC) is known to respond to immune checkpoint blockade (ICB) therapy, whereas there has been limited analysis of T-cell responses to RCC. In this study, we utilized human carbonic anhydrase 9 (hCA9) as a model neoantigen of mouse RENCA RCC. hCA9-expressing RENCA RCC (RENCA/hCA9) cells were rejected in young mice but grew in aged mice. CD8+ T cells were the primary effector cells involved in rejection in young mice, whereas CD4+ T cells participated at the early stage. Screening of a panel of hCA9-derived peptides revealed that mouse CD8+ T cells responded to hCA9288-296 peptide. Mouse CD4+ T cells responded to lysates of RENCA/hCA9, but not RENCA cells, and showed reactivity to hCA9 276-290, which shares three amino acids with hCA9 288-296 peptide. Immunohistochemistry analysis revealed that few T cells infiltrated RENCA/hCA9 tissues in aged mice. ICB therapy of anti-PD-1/anti-CTLA-4 antibodies promoted T-cell infiltration into tumor tissues, whereas no definite antitumor effect was observed. However, additional combination with cyclophosphamide or axitinib, a vascular endothelial growth factor receptor inhibitor, induced complete regression in half of the RENCA/hCA9-bearing aged mice with increased expression of PD-L1 in tumor tissues. These results indicate that hCA9 can be a useful model neoantigen to investigate antitumor T-cell responses in mice with RCC, and that RENCA/hCA9 in aged mice can serve as a non-inflamed 'cold' tumor model facilitating the development of effective combined immunotherapies for RCC.

Transcriptome-wide analysis of intracranial artery in patients with moyamoya disease showing upregulation of immune response, and downregulation of oxidative phosphorylation and DNA repair.

OBJECTIVE: Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive occlusion of the internal carotid artery and the secondary formation of collateral vessels. Patients with MMD have ischemic attacks or intracranial bleeding, but the disease pathophysiology remains unknown. In this study, the authors aimed to identify a gene expression profile specific to the intracranial artery in MMD. METHODS: This was a single-center, prospectively sampled, retrospective cohort study. Microsamples of the middle cerebral artery (MCA) were collected from patients with MMD (n = 11) and from control patients (n = 9). Using microarray techniques, transcriptome-wide analysis was performed. RESULTS: Comparison of MCA gene expression between patients with MMD and control patients detected 62 and 26 genes whose expression was significantly (p < 0.001 and fold change > 2) up- or downregulated, respectively, in the MCA of MMD. Gene set enrichment analysis of genes expressed in the MCA of patients with MMD revealed positive correlations with genes involved in antigen processing and presentation, the dendritic cell pathway, cytokine pathway, and interleukin-12 pathway, and negative correlations with genes involved in oxidative phosphorylation and DNA repair. Microarray analysis was validated by quantitative polymerase chain reaction. CONCLUSIONS: Transcriptome-wide analysis showed upregulation of genes for immune responses and downregulation of genes for DNA repair and oxidative phosphorylation within the intracranial artery of patients with MMD. These findings may represent clues to the pathophysiology of MMD.

Context-Specific Efficacy of Apalutamide Therapy in Preclinical Models of Pten-Deficient Prostate Cancer.

Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents; however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.