Anti-tumor Effect Of Endostatin Combination With SiRNA-CD24 In Mice Burdened Melanoma

BackgroundIt is still difficult for the scientific community to tackle the treatment of melanoma.CD24 is a new immune checkpoint that inhibits macrophage function and promotes tumor progression.Thus,we expect to exert anti-melanoma effects of siRNA-CD24.However,there are many factors that affect tumor progression,and the focus of current cancer treatment has shifted to multitarget therapy.Macrophages could promote tumor growth by secreting VEGF.And Endostatin has been proved that could effectively inhibit the expression of VEGF and play an anti-tumor role.Therefore,this study explored the antimelanoma effect of the co-expression plasmid of siRNA-CD24 and Endostatin,and preliminarily explored its mechanism.ObjectiveTo study whether the co-expression plasmid of siRNA-CD24 and Endostatin could inhibit angiogenesis and improve the anti-tumor immunity of tumor-bearing mice by simultaneously inhibiting the expression of CD24 and VEGF,and significantly exerts the anti-melanoma effect of mice.Methods1.Detection of clinical specimens Immunofluorescence and HE staining were used to detect the difference of CD24 expression and vascular content in melanoma tissues and adjacent normal tissues.2.Construction of the plasmids and functional verification Constructing the siRNA-CD24 interference plasmids.Six-well plate was used to prepare mouse melanoma B16 cells,and siRNA-CD24 plasmids was transfected.Then,the interference of CD24 expression was detected.The co-expression plasmid of pEndostatinsiRNA-CD24 was constructed and transfected into mouse mononuclear macrophages（RAW264.7 cells）to verify its interference effect of the expression of VEGF in these cells.3.Animal experiment The melanoma-bearing mice model was constructed and randomly divided into different groups for different treatments.Changes in subcutaneous tumor size were observed during the treatment.The mice were sacrificed and tumor tissues were separated on the seventh day after the last treatment.Western blot and immunofluorescence was used to detect the expression of CD24,VEGF and CD34 proteins in tumor tissues.The expression of PCNA and Ki67,the infiltration of M1 macrophages and T lymphocytes were detected by immunofluorescence.TUNEL assay was used to evaluate the apoptosis of tumor cells.Hematoxylin-eosin staining was used to detect the contents of blood vessels in tumor tissues.Flow cytometry was used to detect the proportion of T cells and NK cells in the spleen of mice,and the proportion of macrophage subsets in tumor tissues of mice.Results1.In clinical samples,compared with adjacent tissues,CD24 expression was significantly increased in melanoma tissues.2.The self-constructed co-expression plasmid significantly inhibited the expression of CD24 and VEGF in cells.3.Combined treatment significantly inhibited the expression of CD24,VEGF and CD34 in tumor tissue,promoted the apoptosis of tumor cells,inhibited the proliferation of tumor cells,and reduced the angiogenesis in tumor.It significantly inhibited tumor growth and prolonged survival time of tumor-bearing mice.In addition,combination therapy increased the proportion of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor.The infiltration of T lymphocytes in tumor tissue and the proportion of T lymphocytes and NK cells in spleen were significantly increased.ConclusionThe co-expression plasmid of siRNA-CD24 and Endostatin can effectively inhibit the growth of melanoma in mice.Interference of CD24 expression increases the proportion of anti-tumor M1 macrophages.Meanwhile,the anti-tumor immunity of mice was further improved by Endostatin that inhibiting the expression of VEGF to achieve the effect of inhibiting angiogenesis.Therefore,blocking tumor immune target CD24 combined with angiogenesis inhibition therapy may provides a new direction for clinical treatment of melanoma.