| Background and PurposeHepatocelluar carcinoma(HCC)is the second leading cause of cancer-associated death in China owing to its rapid growth,high malignancy and limited treatment.HCC is a highly vascularized tumor with complex tumor microenvironment.Tumor angiogenesis and immunosuppressive tumor microenvironment are responsible for the occurrence and progression of liver cancer.Also they become arrestive issue in liver cancer research,as well as the main targets of systemic therapy for advanced liver cancer at present.In recent years,some clinical trial results of vascular targeted therapy combined with immune checkpoint inhibitor therapy have shown outstanding efficacy,with a high remission rate beyond expectations,bringing great benefits to patients with HCC.However,why vascular targeted therapy combined with immune checkpoint inhibitor therapy has synergistic anti-tumor effect and how to further improve the clinical treatment of hepatocellular carcinoma is an urgent problem to be solved at present.VEGF is the main target of anti-angiogenesis therapy.In this study,we mainly explore the role of VEGF in tumor microenvironment of liver cancer and the mechanism of VEGF regulating the expression of PD-1/PD-L1,as well as evaluating the therapeutic effect of combined VEGF and PD-L1 blockade in mice liver cancer model,so as to provide a new theoretical basis for the clinical treatment of liver cancer.Materials and MethodsIn vitro experiment section1.Immunohistochemical staining was used to detect the expression of VEGF,CD8,CD163,PD-L1 in tumor tissues of patients with hepatocellular carcinoma and to analyze the relationship between the VEGF and the prognosis of patients with hepatocellular carcinoma,as well as the correlation between VEGF and CD8,CD 163,PD-L1.2.To analyze the relationship of VEGF,prognosis of HCC,tumor infiltrating CD8+T cells and macrophages in TCGA database;Gene Ontology Analysis of VEGF Related Genes in TCGA Database3.A co-culture model of activated T lymphocytes and tumor cells in vitro was established to study the effect of VEGF on T cell proliferation and anti-tumor ability by flow cytometry,RT-qPCR,Western blot,Transwell,LDH release test.Also the effect of VEGF on PD-1 expression of T cells and PD-L1 expression of tumor cells respectively,and the mechanism by which PD-1 and PD-L1 were regulated were explored by flow cytometry and western blot.4.Tumor-associated macrophages were induced in vitro to detect the effect of VEGF on the phenotype of tumor-associated macrophages by flow cytometry,Western blot and RT-qPCR,also the effect of VEGF on the expression of PD-L1 in tumor-associated macrophages was analyzed.In vivo experiment sectionThe orthotopic model of HCC in mice was constructed.The mice were treated with PBS,anti-VEGF,antiPD-L1 and anti-VEGF combined with anti-PD-L1,respectively.The survival time,tumor volume,and IHC staining of PCNA were measured to evaluate the therapeutic effect of combined blockade of VEGF and PD-L1.RT-qPCR was used to analyze the expression of PD-1 and IFN-gamma in tumor tissues of each group.The expression of CD8 and PD-L1 in tumor tissues was analyzed by immunohistochemical staining.RT-qPCR was used to analyze the expression of IL-1β,IL-2,IL-4,IL-6,IL-10,TGF-beta,TNF-alpha and IL-12B in tumor tissues of each group.Results1.The expression of VEGF was negatively correlated with the prognosis of HCC patients,and negatively correlated with CD8+T cell infiltration,PD-L1 expression,and positively correlated with macrophage infiltration.2.VEGF inhibited T cell proliferation and anti-tumor ability in vitro,up-regulated PD-1 expression and activated PI3K pathway.VEGF down-regulated the phosphorylation level of STAT3 and the expression of PD-L1 in tumor cells by inhibiting the secretion of IFN-gamma in T cells3.In vitro,VEGF up-regulated the expression of CD 163 and CD206 in tumor-associated macrophages,down-regulated the expression of CD80 and CD86,and indirectly down-regulated the expression of PD-L1 in macrophages.4.In vivo experiment demonstrated that combined VEGF and PD-L1 blockade had synergistic anti-tumor effects,blocking VEGF could up-regulate the expression of PD-L1 and CD8 in tumor tissues and increase the expression of IFN-gamma in tumor tissues.Blocking VEGF and PD-L1 could down-regulate immunosuppressive cytokines and reshape the tumor microenvironment.Conclusion1.VEGF could inhibit T cell function and regulate PD1/PD-L1 expression in tumor microenvironment2.VEGF directly induced tumor-associated macrophages to differentiate into M2-like macrophages,and inhibited Ml-type polarization3.Combined blockade of VEGF and PD-L1 could exert synergistic anti-tumor effects both in vivo and in vitro... |
PDF Full Text Request