The Relevance Between "N2" Polarization Of Tumor Associated Neutrophils And Hepatocellular Carcinoma In Hypoxic Microenvironment

Objective:To investigate the relevance between "N2" polarization of tumor associated neutrophils and Hepatocellular carcinoma in hypoxic microenvironment.Methods:Clinical data of 50 Hepatocellular carcinoma(HCC)patients and 20 hepatic hemangioma patients were collected from the Hepatopancreatobiliary Surgery Department,Zhengzhou University Affiliated Tumor Hospital,as well as paired samples of carcinoma tissues and pericarcinomatous tissue.For a start,to analyze the relevance between neutrophils in peripheral blood and the prognosis in HCC patients,the Receiver Operating Curve(ROC)and the regression model were used to assess the biomarkers.The Kaplan-Meier method was used to calculate survival rate of patients with high or low NLR,and the log-rank test was used for survival analysis?Immunohistochemistry and RT-PCR were used to detect the expression level of "N2" polarization of tumor associated neutrophils(TANs)and HIF-1? in different tissues.The histological localization and expression level of "N2"polarization of TANs and The relevance between "N2" polarization of TANs and HCC were analyzed objectively by the statistic method?Then the following sections is mainly analysis the relevance between "N2" polarization of TANs and HIF-1?in Hepatocellular carcinoma.Finally,hepatoma cell line were co-cultured with neutrophils under hypoxia conditions in vitro.The relevance between "N2"polarization of TANs and HIF-1? was further confirmed by flow cytometry.Results:1.NLR in peripheral blood of HCC patients was significantly higher than that of patients with hepatic hemangioma(p<0.05).There was a significant correlation between NLR and the prognosis of HCC patients(p<0.05).According to ROC Curve and Youden index,the NLR truncation value was 3.67 in these HCC patients(sensitivity 66.67%,specificity 85.71%,p<0.05)?COX regression model muhifactor analysis showed that NLR was a predictor of overall survival(OS)and progression-free survival(PFS)in HCC(p<0.05).There was a significant difference in DFS and OS curve between the two groups with high or low NLR(p<0.05).2.Immunohistochemical staining revealed that "N2" polarization of TANs is widely expressed in carcinoma tissues and pericarcinomatous tissues and normal tissues,but the expressed levels are different:the expression level of "N2"polarization of TANs in carcinoma tissues was higher than that in pericarcinomatous tissues and normal liver tissues(p<0.05)?Survival analysis showed that,compared with negative expression,patients with "N2" polarization of TANs positive expression had a poor progression-free survival(p<0.05),a shorter overall survival(p<0.05).3.The expression level of HIF-1? in carcinoma tissues was higher than that in pericarcinomatous tissues and normal liver tissues(p<0.05);the expression level of HIF-1? was no significant difference between pericarcinomatous tissues and normal liver tissues(p>0.05)?Further analysis shows that there was a positive correlation between the expression level of HIF-1? in carcinoma tissues and the expression level of "N2" polarization of TANs(p<0.05)?Moreover,the interaction between the two had a certain influence on the prognosis of HCC(p<0.05).Furthermore,RT-PCR confirmed the results:there was a positive correlation between the relative quantitative expression of HIF-1? and the relative quantitative expression of ARG1 in carcinoma tissues?4.Neutrophils were cultured in normoxic and hypoxic environments and it was found that the proportion of CD66b positive cells increased in hypoxic environments(p<0.05).Then we establish the co-culture model of hepatocellular carcinoma cell line and neutrophils in vitro.It was found that the proliferation of HCCLM3 in the co-culture group was significantly increased than the control group(p<0.05).At the same time the proportion of CD66b positive cells increased in the co-culture group(p<0.05).Conclusions:1.High NLR showed a negatively correlated with OS and PFS of HCC and NLR is a risk factor for the prognosis of HCC patients.2.The expression level of "N2" polarization of TANs in carcinoma tissues was negatively correlated with the prognosis of HCC patients.3.There was a positive correlation between the expression level of HIF-1? in carcinoma tissues and the expression level of "N2" polarization of TANs.The interaction between the two had a certain influence on the prognosis of HCC.4.For HCC patients,hypoxia microenvironment may influence the numbers of the survival HCCLM3 tumor cells by up-regulating the proliferation of "N2"polarization of TANS.