VEGF Inhibitor Upregulates PD-L1 Expression And Mediates Immunosuppression Of Tumor-associated Macrophages In Ovarian Cancer

Background:Ovarian cancer is one of the three major malignant tumors of the female reproductive system.Although aggressive cytoreductive surgery followed by chemotherapeutic association of platinum and taxanes has benefited patients with advanced ovarian cancer,the 5-year survival rate remains low,hovering around 25-30%.Malignant tumors have entered the era of "precision therapy",and tumor angiogenesis plays an important role in the growth,proliferation,invasion and metastasis of solid malignant tumors.Anti-angiogenesis therapy has been widely used in clinical treatment,however,a large number of clinical trials have shown that anti-angiogenic agents were not effective in the treatment of ovarian cancer and only prolong the progression-free survival of patients.Tumor immune escape is one of the reasons for the poor therapeutic effect.Therefore,our study group intends to confirm the role of VEGFA in the progression of ovarian cancer.To elucidate the changes of immune microenvironment of ovarian cancer after inhibition of VEGFA and to explore its signal transduction mechanism.To investigate the therapeutic effect of PD-1/PD-L1 pathway inhibitor combined with VEGFA inhibitor in ovarian cancer,providing theoretical basis and new therapeutic ideas for clinical transformation.Methods:Immunohistochemical staining was used to analyze the expression of VEGFA/CD68 in ovarian cancer tissue and its correlation with clinicopathological factors as well as the prognosis.Then,the ovarian cancer cell line SKOV3 was externally treated with bevacizumab,and microarray analysis was used to identify differentially expressed genes after bevacizumab treatment.After that,gene functional annotation and pathway enrichment analysis were conducted to find the possible resistance mechanism of bevacizumab.The expression of PD-L1 in ovarian cancer cells and tumor-associated macrophages before and after treated with bevacizumab was measured by Western Blot,QRT-PCR and enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of IFN-γ in cell supernatant after bevacizumab treatment.ShIFNGR1 cell lines were constructed by means of interfering RNA technology and expression vectors to verify the mechanism of bevacizumab to upregulate PD-L1 in ovarian cancer cells.Subcutaneous tumor formation in nude mice demonstrated that the combination of PD-1 inhibitor could enhance the anti-tumor effect of anti-VEGFA inhibitor.Immunohistochemical staining further confirmed the expression of CD31、PD-L1 and F4/80 proteins in tumor tissues.Results:1、The expression of VEGFA/CD68 in ovarian cancer was significantly higher than that in normal ovarian tissue(p=0.0287/0.0343),but was not corrected with overall survival.Analysis of clinicopathological showed that the expression of CD68 was correlated with lymphatic vascular infiltration.2、Microarray analysis results suggested that bevacizumab would activate the tumor regulatory pathways PI3K/AKT and MAPK,which may be related to the mechanism of drug resistance.3、Bevacizumab can up-regulate PD-L1 expression level of tumor cells and PD-1/PD-L1 expression level of TAM,and maybe related to the process of immunosuppression.4、After bevacizumab treatment,the secretion of IFN-γ protein increased in the supernatant of tumor cells and TAM,the expression of IFN-γ receptor(IFNGR1)was also up-regulated,same results were obtained after exogenous addition of recombinant IFN-γ protein.After down-regulated the expression of IFNGR1 by interference technology,and bevacizumab no longer induced up-regulation of PD-L1 expression,suggesting that the mechanism of bevacizumab up-regulate PD-L1 expression may be related to IFN-γ secretion.In addition,recombinant IFN-γ protein can increase the expression of IRF1/NF-κB,which activated the expression of PD-L1.5、Subcutaneous tumor formation in nude mice confirmed that combination of PD-1 inhibitor may enhance the effect of anti-VEGFA inhibitor by modulating the immune microenvironment,reducing the number of TAM and improving the function.Conclusion:1、Bevacizumab increased the secretion of IFN-γ by tumor cells and TAM,which upregulated PD-L1 through IFNGR1-IRF1/NF-κB pathway,and associated with immunosuppression.2、In vivo experiments confirmed that combined with PD-1 inhibitor can reduce the expression of PD-L1 in tumor tissues and TAM infiltration,furthermore enhance the effect of VEGFA inhibitor.