Preliminary Study On Regulatory Mechanism Of Pd-l1 Expression By Macrophages In The Tumor Microenvironment Of Gastric Cancer

Objective:Via a preliminary study on the mechanism by which macrophages regulate PD-L1expression in the microenvironment of gastric cancer,it is possible to provide ideas for the development of effective antitumor immunotherapeutic strategies.Methods:(1)Immunohistochemical staining was used to stain PD-L1,CD3,CD8 and CD68in human gastric cancer tissues.Correlations between PD-L1 expression and CD protein expression,as well as with prognostic values,were statistically analyzed.(2)Western blotting and immunofluorescence staining were used to detect the expression level of endogenous PD-L1 in human gastric cancer cell strains,and the regulatory effect of endogenous PD-L1 expression by differentiated macrophages was verified.Additionally,bioinformatics was used to analyze the macrophage subtypes that were involved in this regulation.(3)An enzyme-linked immunosorbent assay was used to screen the cytokines that may promote PD-L1 expression in SGC cells.Subsequently,Western blotting and immunofluorescence staining were used to verify the expression level of PD-L1 under cytokine stimulation.Finally,cytokine-neutralizing antibodies were used to re-verify the results.(4)Western blotting was used to screen the signaling pathways that were involved in the regulation of PD-L1 expression in SGC cells.Afterwards,signaling pathway inhibitors and cytokine-neutralizing antibodies were used to verify the results.(5)After the overexpression and knockdown of endogenous PD-L1,WST-1 assays and colony formation assays were used to detect the effects of differentiated macrophages on gastric cancer cell proliferation and tumor formation.Results:(1)Both gastric cancer cells and tumor-infiltrating immune cells were observed to express PD-L1(mainly in the cell membrane and cytoplasm).(2)The human gastric cancer cell strain SGC was shown to express endogenous PD-L1,and both M1-type macrophages and M2-type macrophages were able to promote PD-L1 expression in SGC cells,which is more closely related to the polarization function,cytokines and CD proteins of M1-type macrophages.(3)The levels of TNF-αand IL-6 that are secreted by differentiated macrophages were significantly increased,and the expression of PD-L1 was increased.After treatment with TNF-αand IL-6-neutralizing antibodies,PD-L1 expression in the SGC cells was decreased.In the TCGA-STAD database,PD-L1 was positively correlated with TNF-αand IL-6.(4)Both NF-κB and STAT3 inhibitors were shown to inhibit the promotion of PD-L1 expression by differentiated macrophages.After treatment with a TNF-α-neutralizing antibody,the expression levels of p-p65 and PD-L1 in SGC cells were decreased.After treatment with a IL-6-neutralizing antibody,the expression levels of p-STAT3 and PD-L1 in SGC cells were decreased.In the TCGA-STAD database,PD-L1 was positively correlated with p65 and STAT3.(5)After the overexpression of endogenous PD-L1,differentiated macrophages were observed to promote AGS cell proliferation and tumor formation.After the knockdown of endogenous PD-L1,the promoting effect of SGC cell proliferation and tumor formation by differentiated macrophages was weakened.Conclusions:(1)Thirty-five percent of patients were PD-L1 positive in regard to gastric cancer cells.High PD-L1 expression in gastric cancer cells and a low infiltration of CD8+T cells suggest a poor prognosis.(2)The expression level of PD-L1 in gastric cancer cells was positively correlated with the infiltration degree of CD3~+cells,CD8~+T cells and CD68~+macrophages in the tumor microenvironment.In the TCGA-STAD database,PD-L1 was positively correlated with CD3,CD8,and CD68.(3)Differentiated macrophages,which are dominated by M1-type macrophages,promoted PD-L1 expression in human gastric cancer cells by increasing the secretion of the cytokines TNF-αand IL-6;additionally,these macrophages rely on the NF-κB and STAT3 signaling pathways,thus promoting the occurrence and development of gastric cancer.