Studies On The Effects And Mechanisms Of Cyanidin-3-O-glucoside On Colorectal Cancer Mediated By CD169~+ Macrophages

Research background:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.About 1/3 cancer patients die of colorectal cancer every year.Colorectal cancer is the fourth largest fatal cancer in the world,and its morbidity and mortality are also in the third and fifth places in Chinese malignant tumors,which is becoming a worldwide health problem.There is no effective treatment for colorectal cancer by far.It is of great significance to develop new immunotherapy strategies targeting macrophages for clinical treatment of CRC.The development of tumor is regulated by various inflammatory events,including macrophages and their associated factors,especially in digestive tract tumors.Chronic inflammation is one of the key factors inducing colorectal cancer,such as inflammatory bowel disease(IBD).At the same time,the presence of a large number of inflammatory factors in the tumor microenvironment also increases the difficulty of colorectal cancer treatment.CD 169+macrophages is a unique macrophage subset that differs from M1 and M2 macrophages.Some studies have reported that CD169+macrophages play a crucial role in several autoimmune diseases.Our previous findings also showed that CD169+macrophages increased significantly in the colon and lymph nodes in DSS-induced colitis and played a role in exacerbating inflammation in the colon.Therefore,whether CD 169+macrophages are involved in the inflammatory response during the development of cancer is also a matter that we urgently need to determine.Recent studies have shown that anti-inflammatory drugs,such as aspirin and dexamethasone,have great potential in inhibiting tumors by regulating CRC tumor microenvironment.At the same time,Cyanidin-3-O-glucoside(C3G),a phenolic compound with small molecules in various fruits and vegetables,have been proved to have the same anti-inflammatory effect.Our previous study also demonstrated the role of C3G in inhibiting CD169+macrophages to regulate the immune microenvironment in DSS-induced colitis to exert an inhibitory effect on inflammation.Whether C3G can inhibit colorectal cancer is the focus of this study.To clarify the inhibitory effect of C3G on colorectal cancer and to explore whether C3G exerts its anti-tumor effect through CD169+macrophages will provide new drugs and potential targets for the prevention and treatment of colorectal cancer.Research purposes:The purpose of this study was to explore the therapeutic effects and mechanisms of C3G mediated by CD 169+macrophages in colitis-associated colorectal cancer(CAC)animal model induced by AOM/DSS and subcutaneous tumor model of colorectal cancer.At the same time,we aim to explore the role of CD169+ macrophages in colorectal cancer,especially in colitis-associated colorectal cancer,and to explore whether it inhibits the occurrence of colorectal cancer by inhibiting inflammation.Therefore,this study will provide experimental data with theoretical value and clinical application potential for the research and development of new drugs for colorectal cancer,and provide new therapeutic targets and strategy for the prevention and treatment of colorectal cancer.Research methods:CT26 cells and HCT116 cells were treated with 0,1,2,4μM of C3G.CCK8 and Transwell experiments have proved the ability of proliferation and migration of CT26 and HCT116 cells under different doses of C3G treatment.Immunofluorescence and Flow cytometry were used to detect the changes of apoptosis and cell cycle of CT26 cells induced by C3G stimulation.The expression levels of proliferation and apoptosis pathway proteins on colon cancer cells were detected by western blotting analysis.Subcutaneous tumorigenesis model of colon cancer induced by BALB/c-WT mice and CD169-DTR mice with BALB/c background was used to detect the effect of C3G during tumorigenesis.C3G was injected in tumors of mice every 3 days.Various inflammatory factors and immune cells in tumors and spleen were detected by qPCR and Flow cytometric analysis.CD169-DTR mice with C57BL/6 background were also used to induce the AOM/DSS model.One week after AOM injection,mice were given 2.5%DSS solution for seven days,and given water for two more weeks,which was repeated three times to induce inflammatory-associated colorectal cancer models.DT was injected in mice every 3 days during the DSS treatment.And the growth of tumor was observed during this period.H&E staining showed the histopathological changes of colon in different disease stages.qPCR detected the expressed levels of various inflammatory factors.Results:First,the proliferation and migration of colon cancer cells was inhibited directly by C3G in vitro.And the cell cycle of colon cancer cells was blocked by C3G at the G2 phase.C3G induces the apoptosis of colon cancer to suppress proliferation signaling pathways mediated by EGFR by targeting EGFR on cell surfaces.The growth rate of tumor was significantly inhibited in vivo.Meanwhile,the expression of CD 169 and the production of type I IFN were increased due to C3G treatment.And the number of CD8+T cells in tumor and spleen increased.All these results indicated the inhibitory effects of C3G on colon cancer both in vivo and in vitro.Then CD169-DTR mice were used for both subcutaneous tumorigenesis model and AOM/DSS-induced CAC model to explore the involvement of CD169+macrophages during CRC.The tumor growth were inhibited in both models of CD169-DTR with deletion of CD169+macrophages The severity of inflammation and production of inflammatory factors were also decreased significantly in CD169-DTR mice.Moreover,the number of CD8+T cells increased within the tumor.Conclusions:C3G has been proved to have significant inhibitory effect on mouse colon cancer in vivo and in vitro,and its mechanism has been clarified.On the one hand,C3G induced the apoptosis of colon cancer cells by targeting EGFR on tumor cells surface;on the other hand,it regulated tumor immune microenvironment possibly by inhibiting the expression of type I IFN and CD 169.At the same time,tumor growth in was inhibited in CD169-DTR mice with deletion of CD169+macrophages.Similarly,tumorigenesis was inhibited in AOM/DSS induced inflammation-driven colorectal cancer model in CD169-DTR mice,and the colitis was ameliorated in the early phased of CAC model.Therefore,these findings in the present study suggested C3G could be used as a potential drug for the prevention and treatment of colon cancer,and CD169+macrophages can be used as a potential target for the prevention and treatment of colorectal cancer.However,the mechanisms of CD169+macrophages in colorectal cancer needs further study.