Co-inhibition of adenosine 2b receptor and programmed death-ligand 1 promotes the recruitment and cytotoxicity of natural killer cells in oral squamous cell carcinoma.

Adenosine promotes anti-tumor immune responses by modulating the functions of T-cells and natural killer (NK) cells in the tumor microenvironment; however, the role of adenosine receptors in the progression of oral squamous cell carcinoma (OSCC) and its effects on immune checkpoint therapy remain unclear. In this study, we obtained the tumor tissues from 80 OSCC patients admitted at the Shandong University Qilu Hospital between February 2014 and December 2016. Thereafter, we detected the expression of adenosine 2b receptor (A2BR) and programmed death-ligand 1 (PD-L1) using immunohistochemical staining and analyzed the association between their expression in different regions of the tumor tissues, such as tumor nest, border, and paracancer stroma. To determine the role of A2BR in PD-L1 expression, CAL-27 (an OSCC cell line) was treated with BAY60-6583 (an A2BR agonist), and PD-L1 expression was determined using western blot and flow cytometry. Furthermore, CAL-27 was treated with a nuclear transcription factor-kappa B (NF-κ B) inhibitor, PDTC, to determine whether A2BR regulates PD-L1 expression via the NF-κ B signaling pathway. Additionally, a transwell assay was performed to verify the effect of A2BR and PD-L1 on NK cell recruitment. The results of our study demonstrated that A2BR and PD-L1 are co-expressed in OSCC. Moreover, treatment with BAY60-6583 induced PD-L1 expression in the CAL-27 cells, which was partially reduced in cells pretreated with PDTC, suggesting that A2BR agonists induce PD-L1 expression via the induction of the NF-κ B signaling pathway. Furthermore, high A2BR expression in OSCC was associated with lower infiltration of NK cells. Additionally, our results demonstrated that treatment with MRS-1706 (an A2BR inverse agonist) and/or CD274 (a PD-L1-neutralizing antibody) promoted NK cell recruitment and cytotoxicity against OSCC cells. Altogether, our findings highlight the synergistic effect of co-inhibition of A2BR and PD-L1 in the treatment of OSCC via the modulation of NK cell recruitment and cytotoxicity.

Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients.

PURPOSE: The current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. METHODS: A retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. RESULTS: Most OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. CONCLUSIONS: In HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

Whole transcriptome analysis of trophoblasts under hypoxia.

INTRODUCTION: A physiological hypoxia environment exists at maternal-fetal interface during early pregnancy. In addition, there is a pathological hypoxic microenvironment in patients with preeclampsia. Therefore, investigating the hypoxic adaptation and the effects of hypoxia on trophoblasts transcriptome is helpful to better understand the function and regulatory mechanism of trophoblasts at the maternal-fetal interface. METHODS: Trophoblast cell line HTR-8/SVneo was cultured under normoxia and hypoxia for 24 h, the full transcriptome was analyzed via RNA-Seq. GO and KEGG enrichment were performed on differentially expressed mRNA, adjacent genes of differentially expressed lncRNA, host genes of differentially expressed circRNA and target genes of differential expressed miRNA. RESULTS: The results showed that hypoxia differentially regulated 373 mRNAs, 334 lncRNAs, 71 circRNAs and 33 miRNAs. GO and KEGG enrichment showed that hypoxia negatively regulated TLR3 and PI3K-Akt signaling pathways. Consistently, we found hypoxia significantly inhibited TLR3 agonist-induced cytokines expression and the phosphorylation of Akt and mTOR. DISCUSSION: Our study obtained the full transcriptome data and potential regulatory network of trophoblasts under hypoxia, providing supportive data for revealing the function of trophoblasts.

Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients.

Introduction: An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC. Methods: Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored. Results: We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15. Discussion: We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

Interaction Between Macrophage Extracellular Traps and Colon Cancer Cells Promotes Colon Cancer Invasion and Correlates With Unfavorable Prognosis.

Background: Macrophage extracellular traps (METs) and tumor-infiltrating macrophages contribute to the progression of several diseases. But the role of METs and tumor-infiltrating macrophages in colon cancer (CC) has not been illuminated. In this study, we aimed to clarify the prognostic value of METs for CC patients and to explore the interaction between CC cells and METs in vitro and in vivo. Methods: A training cohort consisting of 116 patients and a validation cohort of 94 patients were enrolled in this study. Immunofluorescence (IF) staining was conducted to determine METs formation in CC patients. Cox regression was used to perform prognostic analysis and screen out the best prognostic model. A nomogram was established to predict 5-year overall survival (OS). The correlation between METs with clinicopathological features and inflammatory markers was analyzed. The formation of METs in vitro was detected by SYTOX® green and IF staining, and the effect of METs on CC cells was detected by transwell assays. PAD2-IN-1, a selective inhibitor of peptidylarginine deiminase 2 (PAD2), was introduced to destroy the crosstalk between CC cells and METs in vitro and in vivo. Results: METs levels were higher in CC tissues and were an independent prognostic factor for CC patients. The prognostic model consisting of age, tumors local invasion, lymph node metastasis and METs were confirmed to be consistent and accurate for predicting the 5-year OS of CC patients. Besides, METs were correlated with distant metastasis and inflammation. Through in vitro experiments, we confirmed that there was a positive feedback loop between CC cells and METs, in that METs promoted the invasion of CC cells and CC cells enhanced the production of METs, in turn. This interaction could be blocked by PAD2-IN-1 inhibitors. More importantly, animal experiments revealed that PAD2-IN-1 inhibited METs formation and CC liver metastasis in vivo. Conclusions: METs were the potential biomarker of CC patient prognosis. PAD2-IN-1 inhibited the crosstalk between CC cells and METs in vitro and in vivo, which should be emphasized in CC therapy.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Vitamin E succinate exerts anti-tumour effects on human cervical cancer cells via the CD47-SIRPɑ pathway both in vivo and in vitro.

Vitamin E succinate (RRR-a-tocopheryl succinate, VES) acts as a potent agent for cancer therapy and has no toxic and side effects on normal tissue cells. However, the mechanism by which VES mediates the effects are not yet fully understood. Here, we hypothesised that VES mediates antitumour activity on human cervical cancer cells via the CD47-SIRPɑ pathway in vivo and in vitro. Results indicated that the human cervical cancer HeLa cells treated with VES were more efficiently engulfed by THP-1-derived macrophages. In response to VES, the protein expression of CD47 on cell membranes and the mRNA level of CD47 in different human cervical cancer cells significantly decreased. And the level of calreticulin (CRT) mRNA in the VES-treated cells increased. By contrast, CRT protein expression was not altered. miRNA-155, miRNA-133 and miRNA-326 were up-regulated in the VES-treated HeLa cells. Knocking down miRNA-155 and miRNA-133 by RNA interference increased CD47 protein expression in the VES-treated cells. In vivo efficacy was determined in BALB/C nude mice with HeLa xenografts. Results showed that VES reduced tumour growth, increased overall survival and inhibited CD47 in the tumour transcriptionally and translationally. Furthermore, inflammatory factors (TNF-α, IL-12, IFN-γ, IL-2 and IL-10) in the spleen were altered because of VES treatment. Our results suggest that VES-induced antitumour activity is coupled to the CD47-SIRPɑ pathway in human cervical HeLa cancer cells.

Neutrophils infiltration and its correlation with human papillomavirus status in the oral squamous cell carcinoma.

Purpose: One of the cardinal etiological factors for oral squamous cell carcinoma (OSCC) is Human papillomavirus (HPV). Neutrophils were potential targets of immune therapy for patients with OSCC. The objective of this study was to determine if neutrophils density and HPV status can be used to define a high-risk category of patients in OSCC and to investigate the possible relationship between them. Patients and methods: We performed immunohistochemistry to probe neutrophils infiltration and HPV (P16) expression in 81 patients with OSCC. Prognostic factors for cancer-related survival were evaluated by univariate and multivariate analyses. We used the detection of cytokines to investigate the possible molecular mechanisms between neutrophils infiltration and HPV status. Results: There were significantly higher numbers of CD15+ neutrophils infiltration in OSCC tissues. Higher numbers of CD15+ neutrophils infiltration was related to stage Ⅲ，Ⅳ (p<0.001), poor grade (p<0.001), lymph node metastasis (p=0.014), and the higher preoperative neutrophil-lymphocyte ratio (NLR) (p<0.001). HPV-negative status was also associated with stage Ⅲ，Ⅳ (p=0.001), poor grade (p=0.002), lymph node metastasis (p=0.005), radiotherapy (p=0.038), and the higher NLR (p=0.002). The high density of neutrophils was associated with worse cancer-related survival time (p<0.001) and was an independent prognostic factor for OSCC, while the HPV-positive group was associated with better cancer-related survival time. Moreover, high density of neutrophils was correlated with HPV-negative status in OSCC (p<0.001). Detection of cytokines and chemokines revealed that one of the chemotactic factors of neutrophils, IL-8, was exhibited relatively low expression by HPV-positive OSCC cells, whereas HPV-negative OSCC cells were found to drive an IL-8 secretion profile. Conclusion: Neutrophils infiltration and HPV status appear to be prognostic parameters for OSCC. Overexpression of HPV18 E7 on OSCC cells may participate in depressing neutrophils infiltration to some extent through downregulating expression of IL-8.

Knockdown of c­Myc activates Fas-mediated apoptosis and sensitizes A549 cells to radiation.

Several studies have demonstrated that cancer radiosensitivity is associated with the deregulation of c­Myc, but the relationship between c­Myc and Fas in radioresistance of lung adenocarcinoma remains unclear. In this study, we established radiation-resistant A549 cell model (A549/R), and investigated the roles of c­Myc and Fas in radiation-induced cytotoxicity of A549 cells. Apoptosis detection showed that there were fewer apoptotic cells in A549/R cells treated with radiation than in A549 cells. Western blotting results demonstrated the inverse expression pattern of c­Myc and Fas in A549 and A549/R cells. Suppression of c­Myc expression by small interfering RNA (siRNA) displayed enhancement of Fas-mediated apoptosis in A549/R cells, accompanying a significant decrease of Bid, Bcl­2, pro­caspase­8, -9 and -3 and increase of Bax. In contrast, Fas-mediated apoptosis was attenuated while Fas expression was suppressed by ectopic expression of c­Myc in A549 cells. Moreover, decreased cell viability and increased induction of apoptosis were observed in A549/R cells followed by combinational treatment of c­Myc siRNA and irradiation, whereas, upregulation of c­Myc reduced the sensitivity of A549 cells to irradiation. These results indicated that c­Myc and Fas regulated the sensitivity of A549 cells to irradiation by regulating caspase­8-mediated Bid activation and the subsequent association with the mitochondrial pathway of apoptosis.