Correlation Between Expression Levels Of Tregs Cells And Their Regulators In Cancer Tissues Of ESCC Patients With Prognosis

Objective Esophageal squamous cell carcinoma(ESCC)is the most common pathological type of esophageal cancer in China,the tumor microenvironment plays a crucial role in the occurrence and development of ESCC,regulatory T cells(Tregs)is an important tumor microenvironment immunosuppressive factor,associated with poor prognosis.This study aims to study Tregs cell infiltration in ESCC tumor tissues and cytokines that regulate Tregs cell function,including Glucose-regulated protein 94(GRP94),chemokine ligand18(Chemokine c-c motif ligand 18(CCL18),and angiopoietin 4(Angiopoietin like).The relationship between the expression levels of protein 4,ANGPTL4)and interleukin 6(IL-6)and the prognosis of ESCC patients,and the correlation between Tregs cell infiltration and the above regulatory molecules was discussed.Methods1.Tissue specimens and clinicopathological parameters of patients undergoing radical esophageal cancer resection in Taizhou People’s Hospital from January 2015 to December2018 were collected,and the survival status of patients was followed up by telephone,and the median survival time(mOS)and 3-year overall survival(OS)rates were used as prognostic evaluation indicators;2.The esophageal cancer tissue chip was made by the wax block of the specimen,and the expression levels of GRP94,CCL18,ANGPTL4 and IL-6 proteins were detected by immunohistochemical SP(streptavidin-perosidase).The expression of FoxP3 was detected by immunohistochemistry,and FoxP3 positive was used as the standard for judging Tregs cells,and the invasion of Tregs(FoxP3+)cells in ESCC cancer nests was observed.The number of Tregs cells infiltrated into the cancer nest under each highpower field > 20 was defined as high invasion and 20 ≤ as low invasion;3.The whole cohort was divided into high OS group and low OS group according to median OS,and the clinicopathological parameters,Tregs cell infiltration and expression level of the above regulatory factors were compared between the two groups,and the relevant factors affecting OS were screened out by logistic multivariate analysis;4.The expression levels of Tregs cell infiltration,GRP94,CCL18,ANGPTL4 and IL-6were grouped respectively,and the differences in clinicopathological parameters between the groups were compared.The Kaplan-Meier survival curve was used to estimate the mOS and 3-year OS rates,and the log-rank log rank test was used to compare the differences between groups.COX multivariate analysis was used to screen out independent risk factors affecting OS in ESCC patients;5.Pearson grade correlation analysis was used to study the correlation between the degree of Tregs cell infiltration and the expression of the above regulatory factors,and the potential influencing factors affecting the infiltration of Tregs cells into tumor tissues were discussed,which provided guidance for further discussion of the mechanism of Tregs infiltration.Results1.Finally,105 patients were screened,and the last follow-up time was December 10,2022,and a total of 91 patients died in the whole group and 14 survived.The 3-year OS rate was calculated by taking 36 months after surgery as the node,and by January 06,2021,73 patients had died,32 cases had survived,and the overall 36-month survival rate was 30.5%;2.The expression of GRP94,CCL18,ANGPTL4 and IL-6 proteins in ESCC tissues was detected by IHC method,and the positive rates of the above indexes in ESCC tissues were87%,75%,71% and 85%,respectively.The expression of FoxP3 was detected by IHC method,and the results showed that FoxP3-positive Tregs infiltration could be seen in all ESCC tissues,and there were 70 patients with high infiltration,with a high infiltration rate of 66.67%;3.The whole cohort was divided into high OS group(n=52)and low OS group(n=53)according to median OS(24.5 months),and clinical data showed that there was no significant difference in age,sex,T stage and vascular cancer thrombus between the two groups.There were significant differences in differentiation,N stage,postoperative stage,Tregs infiltration,GRP94 expression,CCL18 expression,ANGPTL4 expression and IL-6 expression between the two groups.Logistic multivariate analysis showed that gender,degree of differentiation,Tregs cell infiltration,GRP94 expression,CCL18 expression,ANGPTL4 expression,and IL-6 expression were independently correlated with the patient’s OS;4.Analyze the correlation between the above meaningful indicators and clinicopathologi cal parameters and their value to the prognosis of patients.The results showed that the e xpression of GRP94 protein and CCL18 factor was significantly different between differ ent differentiation degrees,N stages and postoperative stages.There were significant dif ferences between ANGPTL4 protein in different N stages and postoperative stages.IL-6protein had significant differences between different vascular cancer thrombus,differen tiation degree,N stage and postoperative stage.Tregs had significant differences betwee n different N stages and postoperative stages.The results of Kaplan-Meier survival analy sis showed that the OSCs of GRP94,CCL18,ANGPTL4 and IL-6 were shorter,and the OS of ESCC patients with high Tregs infiltration was worse,and the difference was stati stically significant;5.Using COX multivariate analysis,the results showed that Tregs infiltration,GRP94 expression,CCL18 expression,ANGPTL4 expression and IL-6 expression in ESCC tissues were independent risk factors affecting the OS of patients,and the positive expression of the above indicators indicated that ESCC patients had a poor prognosis;6.Pearson analysis showed that CCL18,ANGPTL4 and IL-6 expression were positively correlated with the degree of Tregs infiltration,and the difference was statistically significant.There was no significant difference between GRP94 expression and Tregs infiltration.Conclusions1.There was a correlation between the positive expression of GRP94 and CCL18 in different differentiation degrees,N stages and postoperative stages.There was a correlation between positive expression of ANGPTL4 in different N stages and postoperative stages.There were correlations between positive expression of IL-6 in different N stages,postoperative stages,vascular cancer thrombus,and differentiation degree.There was a correlation between high infiltration of Tregs in different N stages and postoperative stages.The results of Kaplan-Meier survival analysis showed that patients with high Tregs infiltration and positive expression of the above regulatory factors had shorter OS;2.Tregs infiltration,GRP94 expression,CCL18 expression,ANGPTL4 expression and IL-6 expression in ESCC tissues were independent risk factors affecting the OS of patients;3.Tregs is positively correlated with the expression of CCL18,ANGPTL4 and IL-6.