PD-L1 Mediates TAM/M2 Polarization Regulate Triple Negative Breast Cancer Evolution

Objective:The aim of this research was to explore the role and possible molecular mechanism of PD-L1 in regulating the metastasis and angiogenesis of TNBC cells by regulating the TAM/M2 polarization in vitro.Provide a new theoretical and experimental basis for PD-L1 as a TNBC antitumor metastasis target.Materials and Methods:1.Expression of TAM/M2 surface marker protein CD206 and TAM/M1 surface marker protein CD86 were detected by Flow cytometry,Western blot and Immunofluorescence assays;The secretion of TAM/M2 cytokine IL-10 and TAM/M1 cytokine IL-12 were detected by ELISA assay.2.The conditioned medium was prepared to incubate macrophages with IL-13 and aPD-L1(PD-L1 inhibitor)alone or together.3.The effect of aPD-L1 and the conditioned medium of macrophages proliferation and apoptosis were detected by MTT and Flow cytometry assays.4.The conditioned medium of incubating macrophages with IL-13 and/or aPD-L1 were collected and applied to TNBC cells.The invasion and migration ability were tested by Cell scratch and Transwell assays.5.The conditioned medium of incubating macrophages with IL-13 and/or aPD-L1,and then applied to the TNBC cells.The expression of VEGF and MMP2 were detected by Western blot assay.6.The conditioned medium of incubating macrophages with IL-13 and/or aPD-L1 were collected and applied to HUVEC cells.The angiogenesis ability was detected by Microtubule formation assay.7.TNBC cells were co-cultured with macrophages by Transwell,and the expression of TAM/M2 surface marker protein CD206 and TAM/M1 surface marker protein CD86 were detected by Flow cytometry and Western blot assays;The secretion of TAM/M2 cytokine IL-10 and TAM/M1 cytokine IL-12 were detected by ELISA assay.8.The expression of STAT3 protein and the location of the protein in cells were detected by Western blot and immunofluorescence assays.Results:1.αPD-L1 reverses TAM/M2 polarization.IL-13 induced M2 polarization in RAW264.7 cells,αPD-Ll reversed TAM/M2 polarization,but had no effect on TAM/M1(P<0.05).2.αPD-L1 inhibits TAM/M2 induced TNBC cell invasion and migration:IL-13 induced RAW264.7 conditioned medium significantly promoted the invasion and migration of TNBC cells,and αPD-L1 inhibited its invasion and migration(P<0.05).3.αPD-L1 inhibits TAM/M2 induced TNBC angiogenesis:IL-13 induced RAW264.7 conditioned medium significantly promoted microtubule formation of HUVEC,while αPD-L1 inhibited microtubule formation of HUVEC(P<0.05).IL-13 induced RAW264.7 conditioned medium significantly up-regulated the expression of VEGF and MMP2 in TNBC cells,while αPD-L1 could down-regulate the expression of VEGF and MMP2 in TNBC cells(P<0.05).4.TNBC cells induced TAM/M2 polarization,and positive feedback promoted the malignant evolution of TNBC cells:Co-culture of TNBC cells and TAM indirectly.The results showed that TNBC cells could significantly promote TAM/M2 polarization in RAW264.7 cells,while αPD-L1 could reverse the polarization.The polarization of TAM/M1 was not affected by RAW264.7 co-culture and αPD-L1.5.αPD-L1 regulates TAM/M2 polarization by inhibiting RAW264.7 cell STAT3 phosphorylation to prevent nucleation:IL-13 could significantly promote the expression level of p-STAT3,while the αPD-L1 was given at the same time,the expression of p-STAT3 was significantly inhibited;αPD-L1 prevents IL-13 induced STAT3 phosphorylation and p-STAT3 nucleation.Conclusions:1.αPD-L1 reverses TAM/M2 polarization induced by TNBC cells.2.αPD-L1 inhibits TAM/M2 induced TNBC cells invasion,migration and angiogenesis.3.αPD-L1 regulates TAM/M2 polarization by inhibiting STAT3 phosphorylation and preventing it from entering the nucleus.