PTPROt Regulate M2 Macrophage Polarization In Tumor Microenvironment

Objective:1.To investigate the expression of CD24 molecules in human hepatocellular carcinoma?HCC?,and its effect on dendritic cells?DCs?and T cells activation and differentiation.2.To investigate the expression of protein tyrosine phosphatase receptor-type O truncated?PTPROt?of HCC patients' and healthy volunteers' monocyte/macrophages,and explore its effects on macrophage polarization and the underlying mechanism.Methods:1.CD24 expression of HCC tissues and adjacent tissues were determined by real-time PCR and immunohistochemistry?1HC?;2.Hepal-6-CD24 tumor cells were transformed with the human CD24 gene and stably express the CD24 protein by plasmid transfection,before injected into mouse model to observe whether CD24 molecules can influence the Tumor formation;3.DCs,T cells and macrophages extracted from mice were co-cultured with Hepal-6-CD24 cells,then flow cytometry analysis and enzyme-linked immuno sorbent assay?ELISA?were performed to investigate the effect of CD24 on DCs and T cells activation and differentiation,and the expression of PTPROt on macrophages;4.PTPROt expression of HCC tissues,HCC patients' peripheral blood monocytes and healthy volunteers' peripheral blood monocytes were determined by real-time PCR and IHC,and the difference among them was analysed;5.Macrophages were 3D co-cultured with HCC cell lines,after that,flow cytometry analysis,immunofluorescence staining and ELISA were carried out to determine whether PTPROt can influence macrophage polarization.6.To inhibit c-SRC/JAK2 pathway,JAK2 and c-Src specific inhibitors AZD1480 and Bosutinib were used as well as their shRNAs.Western blotting?WB?,immunofluorescence staining and immunoprecipitation?CO-IP?were performed to explore the interaction among PTPROt,c-SRC and JAK2.Results:1.The expression of CD24 in HCC tissues were significantly higher than adjacent tissues?P<0.05?,and the tumor growth was accelerated in Hepal-6-CD24 administered mice;2.CD24 in HCC cells could activate DCs and decrease the percentage of CD8+and CD4+T cells?P<0.05?;3.When co-cultured with Hepal-6-CD24 cells,macrophages expressed more PTPROt as compared withco-cultured with Hepa 1-6 cells?P<0.05?;4.Compared with healthy volunteers,the expression of PTPROt on HCC patients' peripheral blood monocyte were decreased dramatically?P<0.05?;stronger staining of IL-10 but relatively weaker staining of TNF-awere obtained in HCC tissues of PTPROt^Low group compared to the PTPROH'gh group;5.When cocultured with tumor cells,PTPRO^High group macrophages polarized into M1,PTPROt^Low group macrophages polarized into M2;6.PTPRO block M2 polarization through downregulating c-SRC/JAK2 pathway.Conclusion:1.CD24 could promote HCC development,and inhibit anti-tumor immune responses in tumor microenvironment;2.PTPROt was downregulated in HCC patients' peripheral blood monocytes,that could promote macrophages in tumor microenvironment polarize into M2 and inhibitthe anti-tumor immunity.