The Function And Mechanism Of Different Subsets Of Hepatic Myeloid-derived Suppressor Cells In Tumor Angiogenesis

Myeloid-derived suppressor cells (MDSCs) were a group of immature and heterogeneous population, which could largely amplify in bone marrow, liver, spleen, tumor tissue and peripheral blood of patients and tumor bearing animals with immunosuppressive function, under pathological conditions such as tumor, acute and chronic inflammation and autoimmune, furthermore, the expression and function of MDSCs subsets were also affected by different local microenvironment. Research shows that the progression and prognosis of hepatocellular carcinoma have certain correlation with MDSCs. Therefore, further study on the effects and related mechanism of MDSCs subsets in tumor growth and progression has great significance for the MDSCs targeted treatment of hepatocellular carcinoma.Methods:28days after the establishment of subcutaneous tumor-bearing mice model, Hepatic non parenchymal cells were separated by portal vein perfusion, enzyme digestion and Percoll density centrifugation. Hepatic G-MDSC and Mo-MDSC subset were sorted from Hepatic non parenchymal cells by immunomagnetic beads. The purity of Hepatic G-MDSC and Mo-MDSC subsets were detected by flow cytometry. The morphological characteristics of two subsets were observed by light microscope and electron microscope. The ability that two subsets influence the proliferation of lymphocytes was detected by lymphocyte proliferation assay. The effect of Hepatic G-MDSC and Mo-MDSC subsets on human umbilical vein endothelial cells (HUVEC) into a tube was observed by vessel formation assay. Immunohistochemistry was used to observe the effect of Hepatic G-MDSC and Mo-MDSC on the proliferation of tumor cells and tumor angiogenesis. The factors related angiogenesis in cultured supernatant of Hepatic G-MDSC and Mo-MDSC subsets are detected by antibody chip technology.Results:1. The purity of G-MDSC and Mo-MDSC subsets sorted by the immunomagnetic beads were over85%. Both G-MDSC and Mo-MDSC subsets of tumor-bearing mice were amplified. G-MDSC represent the majority, which were about11.3times amounts than Mo-MDSC subsets.2. Under the light microscope, the majority of Mo-MDSC subsets present round or oval nucleus and less cytoplasm with high nuclear-cytoplasmic ratio, and the nuclear of G-MDSC subset were segmented and cytoplasm was rich, the cells size slightly larger than Mo-MDSC. Observed under the electron microscope, the nuclei of Mo-MDSC subset was round or oval, clear nuclear membrane. There were rough endoplasmic reticulum and less mitochondria. While there were few protrusions on the surface of G-MDSC. The nuclei were lobulated-shape. The mitochondria and rough endoplasmic reticulum could be seen in the cytoplasm, circular secretory granules were also visible.3. Cultured with the culture supernatant of Hepatic Mo-MDSC and G-MDSC subsets, lymphocyte proliferative stimulation index were both significantly lower than the control group, indicating that both two subsets can inhibit T cell proliferation.4. In the vessel formation assay, the tube number of Mo-MDSC group and G-MDSC group are36+2and56+3, respectively. G-MDSC group was significantly higher than Mo-MDSC group. The results showed that G-MDSC subset can significantly promote vessel formation compared with the Mo-MDSC subset.5. There were no significant difference in tumor sizes among each group at3days after subcutaneous inoculation of tumor cells. At7d, The tumor volumes of the Mo-MDSC group and G-MDSC group were slightly larger than the H22group. While14d, The tumor volumes of Mo-MDSC group and the G-MDSC group were significantly bigger than the H22group.6. The results of immunohistochemical staining showed that the positive rate of Mo-MDSC group and G-MDSC group was65.33%and56.23%, respectively, and there was a significant difference comparing with H22group. The microvessel number of G-MDSC group was the largest among three groups, which was also significantly higher than H22group and Mo-MDSC group, but there was no difference between H22group and Mo-MDSC group.7. The results of antibody microarray showed that G-MDSC subset could secrete high levels of MCP-lcompared with Mo-MDSC subset. MCP-1was a powerful pro-angiogenic factor, which could promote angiogenesis.8. When MCP-1 antibodies were added, the number of tube of HUVEC was significantly reduced.Conclusion:1. In tumor-bearing mice, Both hepatic G-MDSC and Mo-MDSC subsets were amplified, while G-MDSC subset represent the majority.2. Both hepatic G-MDSC and Mo-MDSC subsets were able to inhibit the proliferation of T lymphocytes.3. Both hepatic G-MDSC and Mo-MDSC subsets were able to promote proliferation of tumor cells.4. Compared with Mo-MDSC subset, G-MDSC subset could significantly promote vessel formation and tumor angiogenesis.5. Hepatic G-MDSC subset probably promote tumor angiogenesis by MCP-1.