Mechanisms Of FFCT On Inducing Autophagy Of Colon Cancer Cells And Promoting Polarization Of Tumor-associated Macrophages

BackgroundColorectal cancer(CRC)is the third most common and fourth highest mortality tumor in the world,and its incidence has increased year by year.It is a serious threat to human life and health.Therefore,it is of great significance to find drugs that can further improve the treatment of colorectal cancer.Extracellular vesicles carry proteins,mRNA,and a variety of intracellular active ingredients,which are important mediators of cell-to-cell communication and participate in the spread of intercellular biological signals.The pathological and physiological effects of extracellular vesicles in tumors have received increasing attention and may serve as potential new targets for tumor treatment.Autophagy is a ubiquitous physiological phenomenon of cells and plays an important role in maintaining the survival,differentiation and tissue development of cells.It also plays an important role in the occurrence and development of tumors.The cytoplasmic components and organelles designated for elimination are surrounded in double-membrane vesicles(autophagosomes),which partly fuse with lysosomes,thus forming autolysosomes.The contents of the autolysosomes are decomposed for recycling to realize the needs of cell metabolism,energy cycle,organelle renewal and intracellular defense.The other part of the autophagosomes is transported to the outside of the cell,as a class of extracellular vesicles,which carry a large amount of cellular information substances.Macrophages in tumor tissues are called tumor-associated macrophages(TAMs),are the most abundant immune cell population present in tumor tissue.Many functions have been attributed to macrophages during tumor growth and progression,including promotion of tumor cell invasion and metastasis,angiogenesis,lymphangiogenesis and immune suppression.Therefore,targeted modulation the polarization of TAMs from pro-tumor alternative activation type(M2)to anti-tumor classical activation type(M1)may be an important antitumor therapeutic strategy.Considering the natural phagocytic ability of macrophages,the study of TAMs recognizing and uptaking of extracellular vesicles secreted by tumor cells has become one of the hot spots in tumor immunology research.Recently,the auxiliary role of Chinese medicine in the prevention and treatment of colorectal cancer has attracted more and more attention.FFCT,anempirical prescription for the treatment of colorectal cancer developed by Jiangsu Province Hospital on Integration of Chinese and Western Medicine,has a certain anti-tumor effect.The specific anti-tumor mechanism of FFCT is not clear yet.Our previous studies showed that FFCT can induce autophagy in colon cancer cells.In view of the close relationship between tumor cell autophagy,autophagosomes and TAMs,to investigate whether autophagosomes produced by colon cancer cells treated by FFCT can polarize TAMs and suppress colon cancer cells growth has great significance for the study of the anti-tumor mechanism of traditional Chinese medicine.Objective1.Preparation and quality control of FFCT and to study the effect of FFCT on autophagy in colon cancer cells.2.To investigate the effect of autophagosomes induced by FFCT on the mice macrophage polarization.3.To study the effect of FFCT on colon tumor growth and TAMs through inducing autophagy and producing autophagosomes in vivo.Methods:1.The quality control of FFCT was conducted by HPLC-DAD.2.The effects ofFFCT on autophagy and autophagosomes secretion of colon cancer cells were observed by fluorescence microscope and electron microscope.The expression of autophagy marker protein LC3? was identified by western blotting.3.Laser scanning confocal microscope was used to detect the recognition and uptake of autophagosomes secreted by colon cancer cells after treated with FFCT in mice macrophage.Flow cytometry,RT-qPCR and ELISA were used toexamine the effects of autophagosomes on mice macrophage polarization.4.The effects of FFCT oncolon cancer cells autophagy and TAMs polarization in vivo were observed by colon cancer subcutaneous xenograft model.Results:1.The main ingredients of FFCT are including Astragaloside A,Oleanic acid,Uosolic Acid,Ginsenoside Rgl,Matrine,Ginsenoside Rb1,Triolein and Eoixol(0.101mg/g,1.107mg/g,0.148mg/g,0.236mg/g,0.056mg/g,0.249mg/g,0.407mg/g and 0.200mg/g,respectively).In the FFCT group,the proliferation of CT26.WT cells were significantly reduced and ina dose-dependent manner within a certain concentration(P<0.0001).The FFCT in 10 mg/ml had no significant effect on the growth of CT26.WT(P=0.3038).2.In the FFCT group,positive staining was observed around the nucleus of eGFP-LC3 CT26.WT cells,and LC3 aggregation to form autophagosomes,which similar to the positive control group(rapamycin);the expression of LC3II increased in a dose-dependent manner;more autophagosomes were clearly observed in the colon cancer cells under the electron microscope;the vesicles extracted from the supernatant of cell culturehad double-membrane structures and the features were similar to autophagosomes.Western blotting confirmed that the vesicles contained a lot of LC3II.3.After the macrophages of mice were co-cultured with autophagosomes secreted by colon cancer cells induced by FFCT,the macrophages were found to be able to efficiently uptake the DiI-labeled autophagosomes and the volume were significantly increased.There were irregular vesicles in the cytoplasm and more pseudopodia were formed.The expressions of related molecules CD86,CD80,TLR2,TLR4,and MHCII were significantly upregulated.The expression levels of M1-related genes were significantly elevated,such as iNOS,TNF-a,MCP-land IL-6.While expression level of M2-related genes was significantly reduced,such as CD206,IL-10,Arg-1 and FIZZ-1.The expression of TNF-aand IL-6 in the supernatant of culture was significantly increased.In addition,the expression of TNF-aand IL-6 in the supernatant of culture were significantly increased.4.The body weights of the mice were not significantly affected by FFCT(P=0.0524).Compared with the control group,the tumor volume and mass were significantly reduced(P<0.0001,P=0.0008)and the number of M1 macrophages in the tumor tissue was significantly higher in the FFCT group.Conclusion:FFCT induced autophagy of colon cancer cells and secreted autophagosomes,whichcould be recognized and taken up by macrophages.Autophagosomes secreted by colon cancer cells reduced by FFCT could promote macrophage to M1 polarization.This study initially revealed themechanism of FFCT against colorectal cancer and provided the experimental basis for further research.