CD177 drives the transendothelial migration of Treg cells enriched in human colorectal cancer.

Objectives: Regulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness. Methods: Single-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments. Results: ScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro. Conclusions: These results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.

High-level of intratumoral GITR+ CD4 T cells associate with poor prognosis in gastric cancer.

Immunotherapy targeting glucocorticoid-induced TNFR-related protein (GITR) exhibited strong anti-tumor capacity in mouse model but poor efficacy in clinical trials. This may be attributed to the different GITR expression mode between human and mouse. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data of human gastric cancer (GC) and used flow to explore the GITR expression across T cell subsets and tissue types in GC patients. We revealed that GITR+ CD4 T cells, including regulatory CD4 T (Treg) cells and conventional CD4 T (Tconv) cells, might contribute to the immunosuppressive microenvironment in GC. The enrichment of these cells was associated with a worse prognosis. Moreover, we found the cellular distribution of GITR protein in Treg cells was microenvironment dependent. In conclusion, GITR is still an important immune checkpoint need to be studied.

Neutrophil extracellular traps promote metastasis in gastric cancer patients with postoperative abdominal infectious complications.

Postoperative abdominal infectious complication (AIC) is associated with metastasis in locally advanced gastric cancer (GC) patients after radical gastrectomy. However, the underlying mechanism remains unclear. Herein, we report that neutrophil extracellular traps (NETs), the DNA meshes released by neutrophils in response to infection, could promote GC cells proliferation, invasion, migration and epithelial-mesenchymal transition dependent on TGF-ß signaling. Then we model nude mice with cecal puncture without ligation to simulate postoperative AIC and find that NETs in peripheral blood and ascites fluid facilitate GC cells extravasation and implantation into liver and peritoneum for proliferation and metastasis. Notably, TGF-ß signaling inhibitor LY 2157299 could effectively impede liver and peritoneal metastasis but not concurrently aggravate sepsis in those AIC-bearing nude mice. These findings implicate that targeting downstream effectors of NETs such as TGF-ß signaling might provide potential therapeutic prospect to reduce the risk of GC metastasis.

Long Non-Coding RNA NRON promotes Tumor Proliferation by regulating ALKBH5 and Nanog in Gastric Cancer.

Long non-coding RNAs (lncRNAs) act as tumor suppressors or oncogenes in tumor development and progression. In this study, we explored the expression and biological role of lncRNA NRON in gastric cancer (GC). We observed that lncNRON was upregulated in GC tissues and cell lines, and high lncNRON expression was associated with malignant features and poor prognosis in GC patients. LncNRON was found to promote the proliferation and tumorigenicity of GC cells. Mechanistically, lncNRON exerted its oncogenic functions by binding to the N6-methyladenosine eraser ALKHB5 and mediating Nanog mRNA decay. In conclusion, our results suggest that lncNRON serves as an oncogenic lncRNA in GC and thus may be a promising prognostic factor and potential therapeutic target for GC patients.

Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia.

Bile acid reflux and subsequent caudal-related homeobox 2 (CDX2) activation contribute to gastric intestinal metaplasia (IM), a precursor of gastric cancer; however, the mechanism underlying this phenomenon is unclear. Here, we demonstrate that alkylation repair homolog protein 5 (ALKBH5), a major RNA N6-adenosine demethylase, is required for bile acid-induced gastric IM. Mechanistically, we revealed the N6-methyladenosine (m6A) modification profile in gastric IM for the first time and identified ZNF333 as a novel m6A target of ALKBH5. ALKBH5 was shown to demethylate ZNF333 mRNA, leading to enhanced ZNF333 expression by abolishing m6A-YTHDF2-dependent mRNA degradation. In addition, ALKBH5 activated CDX2 and downstream intestinal markers by targeting the ZNF333/CYLD axis and activating NF-κB signaling. Reciprocally, p65, the key transcription factor of the canonical NF-κB pathway, enhanced the transcription activity of ALKBH5 in the nucleus, thus forming a positive feedforward circuit. Furthermore, ALKBH5 levels were positively correlated with ZNF333 and CDX2 levels in IM tissues, indicating significant clinical relevance. Collectively, our findings suggest that an m6A modification-associated positive feedforward loop between ALKBH5 and NF-κB signaling is involved in generating the IM phenotype of gastric epithelial cells. Targeting the ALKBH5/ZNF333/CYLD/CDX2 axis may be a useful therapeutic strategy for gastric IM in patients with bile regurgitation.

Norepinephrine Enhances Aerobic Glycolysis and May Act as a Predictive Factor for Immunotherapy in Gastric Cancer.

METHODS: Monoamine neurotransmitters were detected in gastric cancer tissue and paired normal tissue, and The Cancer Genome Atlas was used to identify differentially expressed norepinephrine-degrading and synthetic enzymes. Quantitative real-time PCR and the Seahorse assay were used to determine the effect of norepinephrine on gastric cancer cell glycolysis. MAOA expression in cancer tissues was analyzed by immunohistochemistry and was compared with the patient SUVmax value of PET-CT and other clinicopathological characteristics. RESULTS: The norepinephrine levels were markedly high in gastric cancer tissue, while the norepinephrine-degrading enzymes MAOA and MAOB showed low expression. High norepinephrine levels were associated with activated glycolysis. The MAOA or MAOB expression levels in tumor tissue were closely correlated with the patient SUV max values of PET-CT and immunotherapy evaluation indices, such as PD-L1 and the microsatellite status. CONCLUSIONS: Norepinephrine shows relatively higher expression in gastric cancer tissue than in normal tissue, and its expression level is associated with the glycolysis levels in patients. The norepinephrine-degrading enzymes MAOA and MAOB have significant expression differences in cancer and normal tissue, and their missing or low expression may predict immune therapy outcomes for gastric cancer patients. High norepinephrine levels with metabolic abnormalities may be more suitable for metabolic targeted therapy or immunotherapy.

Long noncoding RNA PVT1 facilitates high glucose-induced cardiomyocyte death through the miR-23a-3p/CASP10 axis.

Dilated cardiomyopathy (DCM) is the leading cause of morbidity and mortality in diabetic patients. Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been shown to be related to the pathogenesis of DCM. However, the mechanism by which PVT1 regulates DCM pathogenesis is unclear. High glucose level was employed to construct a DCM cell model in vitro. Cell viability was determined via cell counting kit-8 assay. The level of lactate dehydrogenase (LDH) was measured with the corresponding kit. Expression levels of PVT1, miR-23a-3p, and caspase-10 (CASP10) messenger RNA were evaluated with a quantitative real-time polymerase chain reaction. Cell apoptosis was assessed by flow cytometry assay. Protein levels of B-cell lymphoma 2-associated X (Bax), cleaved-caspase-3 (cleaved-casp-3), and CASP10 were examined via western blot analysis. The relationship between PVT1 or CASP10 and miR-23a-3p was verified with dual-luciferase reporter assay. We observed that PVT1 and CASP10 were upregulated while miR-23a-3p was downregulated in high glucose-induced cardiomyocytes. High glucose levels repressed cardiomyocyte activity and induced cardiomyocyte apoptosis, but this influence was antagonized by PVT1 knockdown or miR-23a-3p overexpression. Furthermore, PVT1 acted as a sponge for miR-23a-3p, and miR-23a-3p inhibition counterbalanced the influence of PVT1 silencing on viability and apoptosis of cardiomyocytes under high glucose level treatment. PVT1 could increase CASP10 expression via sponging miR-23a-3p. In conclusion, PVT1 acted as a deleterious lncRNA in DCM. PVT1 facilitated cardiomyocyte death by regulating the miR-23a-3p/CASP10, which offered a new mechanism to comprehend the pathogenesis of DCM.

Hypoxic gastric cancer-derived exosomes promote progression and metastasis via MiR-301a-3p/PHD3/HIF-1α positive feedback loop.

Hypoxic tumor microenvironment(TME) is a universal feature in solid carcinoma and is associated with unfavorable prognosis. Tumor-derived exosomes are now significantly implicating in mediating cellular communication and interactions in TME. The aim of this study was to identify exosomal miR-301a-3p involved in gastric cancer(GC) progression and metastasis. Here, we found hypoxia promote GC exosomes release and miR-301a-3p expression in an HIF-1α-dependent manner. In hypoxic TME, enriched miR-301a-3p could be transmitted between GC cells via exosomes and then contributed to inhibit HIF-1α degradation through targeting PHD3, that were capable to hydroxylate HIF-1α subunits to ubiquitinate degradation. This synergistical positive feedback loop between HIF-1α and miR-301a-3p facilitated GC proliferation, invasion, migration, and epithelial-mesenchymal transition. In clinical samples, we further discovered circulating exosomal miR-301a-3p in serum was positively related with peritoneal metastasis. Collectively, these data indicate that GC cells could generate miR-301a-3p-rich exosomes in the hypoxic TME, which then help to HIF-1α accumulation and promote GC malignant behaviors and metastasis. Exosomal miR-301a-3p/HIF-1α signaling axis may serve as a promising predictor and potential therapeutic target of GC with metastasis.

Postoperative quality of life after laparoscopy-assisted pylorus-preserving gastrectomy compared with laparoscopy-assisted distal gastrectomy for early gastric cancer.

BACKGROUND AND AIM: This study aimed to investigate the postoperative quality of life (QOL) between laparoscopy-assisted pylorus-preserving gastrectomy (LAPPG) and laparoscopy-assisted distal gastrectomy with Billroth I anastomosis (LADGBI) in patients with middle-third early gastric cancers (EGC). METHODS: From January 2015 to August 2017, a total 91 patients with EGC underwent LAPPG or LADGBI procedure with complete QLQ-C30 and QLQ-STO22 in Ren Ji Hospital. Not only do surgical and oncological safety as well as clinicopathologic characteristics analyze, but also chronological changes of QOL and nutritional status were compared for the evaluation of functional advantages. RESULTS: There was no significant difference in clinicopathologic characteristics and perioperative recovery between LAPPG and LADGBI. During postoperative 2-year follow up, LAPPG showed significant advantages over LADGBI in emotional functioning, insomnia, appetite loss, reflux, and taste problem and presented larger area in global and functional scales and less area in symptom scales, which means that LAPPG tended to present better improvement and less symptoms than LADGBI for QOL 2 years after surgery. Furthermore, LAPPG could bring about significant improvement in total protein and hemoglobin compared to LADGBI at postoperative 2 years. CONCLUSION: LAPPG obtains QOL as well as total protein and hemoglobin superiority and could be recommended to patients with EGC whose tumor located in the middle third of the stomach.

Prognostic significance of postoperative complication after curative resection for patients with gastric cancer.

PURPOSES: To investigate the impact of postoperative complication on long-term survival in gastric cancer patients after curative resection. METHODS: A total 663 gastric cancer patients undergoing potentially curative resection during January 2010 to November 2014 were studied. Complications were classified according to the Clavien-Dindo severity classification. RESULTS: Postoperative complications occurred in 138 of 663 cases (20.8%). Multivariate analysis identified >65 years old (P = 0.001), male (P = 0.035), and total gastrectomy (P = 0.037) as independent risk factors for postoperative complications. The 5-year overall survival rate was 48.7% in the no complication group, which was significantly better than that in the complication group (42.7%, P = 0.01). Further stratified analysis by cancer staging revealed that decline in 5-year overall survival due to postoperative complication existed among stage I (P = 0.032), II (P = 0.041), and III (P = 0.001) patients. Cox proportional hazards model showed that increasing pT (P = 0.013) and pN (P = 0.001) grade, advanced pTNM (P = 0.001) stage, total gastrectomy (P = 0.001), and postoperative complication (P = 0.006) were independent prognostic factors. CONCLUSION: Postoperative complications have a significant negative impact on the long-term survival in gastric cancer patients even if the tumor is resected curatively.

The RNA-Binding Protein DDX18 Promotes Gastric Cancer by Affecting the Maturation of MicroRNA-21.

OBJECTIVES: The noncoding RNAs (ncRNAs) play important roles in gastric cancer. Most studies have focused on the functions and influence of ncRNAs, but seldom on their maturation. DEAD box genes are a family of RNA-binding proteins that may influence the development of ncRNAs, which attracted our attention. By combining a small sample for high-throughput gene microarray screening with large samples of The Cancer Genome Atlas (TCGA) data and our cohort, we aimed to find some gastric cancer-related genes. We evaluated the clinical significance and prognostic value of candidate gene DDX18, which is overexpressed in gastric cancer tissues. To provide a theoretical basis for the development of new therapeutic targets for the treatment of gastric cancer, we investigated its effect on the malignant biological behavior of gastric cancer in vitro and in vivo, and also discuss its mechanism of action. METHODS: (i) The differential profiling of mRNA expression in five pairs of gastric cancer and adjacent normal tissues was studied by Arraystar Human mRNA Microarray. By combining this with TCGA data and our cohort, we finally filtered out DDX18, which was upregulated in gastric cancer tissues, for further investigation. (ii) The protein expression of DDX18 was detected by immunohistochemistry staining. Then the relationship between the DDX18 expression level and the clinicopathological data and prognosis was analyzed. (iii) A CCK-8 assay and colony formation assay were used to evaluate the effect of DDX18 on cell growth and proliferation in vitro. A transwell assay was also performed to examine the migration and invasion of gastric cancer cells. Cell apoptosis was analyzed by using a fluorescein isothiocyanate-annexin V/propidium iodide double-staining assay. To identify the role of DDX18 in the tumorigenic ability of gastric cancer cells in vivo, we also established a subcutaneous gastric cancer xenograft model. Coimmunoprecipitation, small RNAseq, and western blotting were performed to explore the mechanism of action of DDX18 in gastric cancer. A patient-derived xenograft (PDX) model was used to confirm the effect of DDX18 in gastric cancer tissues. RESULT: (i) DDX18 was upregulated in gastric cancer tumor tissues from a TCGA database and our cohort. The expression of DDX18 was also closely related to tumor volume, Borrmann classification, degree of tumor differentiation, cancer embolus, lymph node metastasis, and TNM stage. (ii) DDX18 could promote cell proliferation, migration, and invasion and inhibit cell apoptosis in vivo and in vitro. (iii) DDX18 could promote the maturation of microRNA-21 through direct interaction with Drosha, decreasing PTEN, which could upregulate the AKT signaling pathway. (iv) The PDX model showed that DDX18 could promote the proliferation of gastric cancer tissues by means of the PTEN-AKT signaling pathway. CONCLUSIONS: (i) DDX18 can be treated as a molecular marker to assess the prognosis of patients with gastric cancer. (ii) DDX18 could be a potential therapeutic target in gastric cancer.

Objective evaluation of clinical outcomes of laparoscopy-assisted pylorus-preserving gastrectomy for middle-third early gastric cancer.

BACKGROUND: Laparoscopic-assisted pylorus-preserving gastrectomy (LAPPG) is a minimally invasive function-preserving surgery for early gastric cancer. This study was designed to investigate the clinical outcomes between LAPPG and laparoscopy-assisted distal gastrectomy (LADG) by objective evaluation. METHODS: A total 167 pT1N0M0 gastric cancer patients underwent LAPPG(n = 70) and LADG(n = 97) were retrospectively analyzed. By evaluating the functional advantages, objective short-term and one year follow-up outcomes were compared. RESULTS: There is no significant difference in perioperative clinical characteristics as well as pathologic results between LAPPG and LADG group while the cost is higher in latter(p = 0.004). The Clavien-Dindo grade II or higher complications were 15.7 and 13.4% in LAPPG and LADG group respectively(p = 0.824). In one year follow-up, nutritional status was significantly better in LAPPG group accompanied by better pylorus function preserving. CONCLUSION: LAPPG is an acceptable surgical procedure for pT1N0M0 middle portion gastric cancer patients in terms of nutritional and economic advantage. TRIAL REGISTRATION: Chinese Clinical Trial Registry ( ChiCTR-PIC-17012358 , Date of Registration:2017-08-14).

Comparison of postoperative lymphocytes and interleukins between laparoscopy-assisted and open radical gastrectomy for early gastric cancer.

OBJECTIVE: This study aimed to study the effects of laparoscopic-assisted radical gastrectomy (LAG) and open radical gastrectomy (OG) on immune function and inflammatory factors in patients with early gastric cancer. METHODS: Seventy-five patients with pT1N0M0 gastric cancer in Ren Ji Hospital from August 2017 to January 2018 were studied. Lymphocytes subsets and interleukins were compared preoperatively and on the third postoperative day (POD3) and seventh postoperative day (POD7). RESULTS: There were no significant differences in age, sex, body mass index, duration of the operation, estimated blood loss, total gastrectomy rate, postoperative first fluid diet, and the levels of preoperative lymphocytes subsets and interleukins between the two groups. The number of CD4+ T cells and the CD4+/CD8+ ratio in the LAG group were significantly higher than those in the OG group on POD3. However, the number of CD8+ T cells, and interleukin-6 and interleukin-8 levels in the LAG group were significantly lower than those in the OG group on POD3. CONCLUSIONS: Laparoscopy can effectively reduce the levels of inflammatory factors and has less effect on the immune system than OG.

miR-182 controls cell growth in gastrointestinal stromal tumors by negatively regulating CYLD expression.

Gastrointestinal stromal tumors (GISTs) are the most common type of mesenchymal tumor of the digestive tract. MicroRNAs (miRNAs) are short non-coding RNAs, which control gene expression at a post-transcriptional level. Dysregulated miRNAs are involved in various types of human disease, including cancer. In the present study, it was revealed that miRNA-182 (miR-182) expression was significantly upregulated in human GISTs compared with adjacent normal tissues. Overexpression of miR-182 enhanced GIST-T1 cell growth, with increased proliferation and decreased apoptosis. miR-182 upregulation also promoted colony formation and migration of GIST-T1 cells. In addition, cylindromatosis (CYLD) was identified as a direct target of miR-182. Overexpression of miR-182 suppressed CYLD expression and enhanced downstream nuclear factor (NF)-κB activation. It was also determined that the expression of CYLD was downregulated in association with upregulated miR-182 in human GISTs. In conclusion, these results demonstrated that miR-182 promoted GIST cell growth by negatively regulating CYLD expression. These findings indicated that miR-182 antagonist may be a promising therapeutic strategy for the treatment of human GIST.

Is 72 h of antimicrobial prophylaxis better than 24 h in elective gastric cancer surgery?

Background/aim: The optimum duration of antimicrobial prophylaxis in elective gastric cancer surgery is still not yet established. The aim of this study is to evaluate the efficacy of 24 h or 72 h of antimicrobial prophylaxis for preventing postoperative infection. Materials and methods: Between July 2016 and January 2018, 990 gastric cancer patients undergoing surgery with D2 lymphadenectomy in Ren Ji Hospital were classified into 24-h or 72-h antimicrobial prophylaxis groups. The incidence of postoperative infection complications was compared. Results: A total of 990 patients (24-h antimicrobial prophylaxis, 708 cases; 72-h antimicrobial prophylaxis, 282 cases) were analyzed. Surgical site infection (SSI) occurred in 37 patients (5.2%) in the 24-h group and 17 patients (6.0%) in the 72-h group, respectively, and 24-h antimicrobial prophylaxis was not a risk factor for remote infection (11.2% in 24-h versus 10.2% in 72-h group). Age >60 years and pathological stage III were significantly associated with remote infection. Conclusion: Compared to 72 h of antimicrobial prophylaxis, 24 h is not a risk factor for either SSI or remote infection. Extended antimicr obial prophylaxis might decrease remote infections for older patients or those of pathological stage III.

[Application of laparoscopic function-preservation proximal gastrectomy in the treatment of early gastric cancer].

OBJECTIVE: To discuss the safety and feasibility in the preservation to hepatic branch of vagus nerve by the side-to-side tubular gastroesophageal anastomosis within the laparoscopic radical proximal gastrectomy for early gastric cancer(EGC). METHODS: Retrospective analysis on the intraoperative and postoperative data of 7 EGC patients receiving laparoscopic radical proximal gastrectomy from January 2014 to January 2015 was carried out. All the patients underwent the preservation of hepatic branch of the vagus nerve by side-to-side tubular gastroesophagreal anastomosis. RESULTS: All the 7 patients completed operations successfully without conversion to open surgery. The mean operative time was (213.1 ± 22.1) minute, the mean reconstruction time was (56.9 ± 11.6) minute, and the mean blood loss was (38.6 ± 28.1) ml. Postoperative time to flatus was (2.4 ± 0.5) day, and postoperative hospital stay was (9.3 ± 0.9) day. No operation-related complications were observed. No severe malnutrition, no recurrence or death, and no severe esophageal reflux during follow-up period were found. CONCLUSION: The preservation of hepatic branch of the vagus nerve by side-to-side tubular gastroesophagreal anastomosis within laparoscopic radical proximal gastrectomy for ECG is safe and feasible.

[Function preserving gastrectomy].

Under the premise of radical resection in the treatment, it is of great significance to preserve partial gastric function so that the early gastric cancer (EGC) patients' postoperative quality of life (QOL) can be improved. In the patients with EGC in the upper third of the stomach, the emphasis is on the prevention of reflux esophagitis caused by bile and gastric juice reflux. Pylorus-preserving gastrectomy (PPG) is applicable to the patients with EGC in the middle third of the stomach. In the patients with EGC in the lower third of the stomach, distal gastrectomy (DG) is performed in general. Various anastomosis ways are applied to reduce the negative impact of pylorus resection after DG. Furthermore, it should also be considered that reasonable vagal nerves preservation and lymph node dissection are both important for function preserving gastrectomy of EGC. Rational use of laparoscopy-assisted gastrectomy has advantages of lower invasiveness, faster recovery, etc. And the amplification effect of laparoscope can contribute to preserving nerves and gastric function.