Isolation, Identification And Immunological Properties Of Exosomes Derived From Colorectal Cancer Stem Cells

Colorectal cancer is a common gastrointestinal malignant tumor, its morbidity and mortality are in third place and second place in Western countries. Currently, the main way to treat colorectal cancer is surgical resection combined with chemotherapy or reflected rays therapy. However, this method of treatment can control the progression of the disease, it can not completely cure the disease. Therefore, to explore the new method of treatment for colorectal cancer becomes a scientific problem to be solved in this area.Cancer stem cells(CSCs) and adult stem cells have a similar ability to self-renewal and differentiation. The cancer stem cells hypothesis believed that the CSCs are the foundation of tumorigenesis, metastasis and recurrence. Colorectal cancer stem cells(CR-CSCs) as the research target provide a new idea for the treatment of colorectal cancer. Exosomes(EXO) which are secreted by cells have a double membrane structure, and the components of protein have a high degree of similarity with maternal cells. The EXO derived from tumor cells will carry tumor surface antigens.In recent years, the research about tumor cells derived EXO as a vaccine has been widely reported. However, the immunity research about CSCs derived EXO has been rarely reported. Tumor cells derived EXO itself has low immunogenicity, how to improve the immunogenicity of EXO becomes the difficult problem of this area. So,improving the immunogenicity of EXO derived from CR-CSCs has great significance for the prevention and treatment of colorectal cancer.In this project, CR-CSCs were separated from cultured CT-26 cell line by magnetic activated cell sorting(MACS), and identify the morphology, phenotype and pluripotency of CR-CSCs through morphological observation, specific marker detection and pluripotency genes expression. EXO precipitant was used to isolate EXO from normal CR-CSCs culture supernatant and heat shock supernatant, andidentify the morphology and specific markers of EXO through scanning electron microscopy(SEM) and specific marker detection. The influence of EXO on peripheral blood mononuclear cells(PBMCs) proliferation, secretion and killing capacity was analyzed through co-culture experiment of EXO and PBMCs, and the expression of immune related molecules was detected by immunoblotting. Finally, in vivo and in vitro experiments were used to evaluate the function of immune preventive function and immune therapeutic function.In vitro results showed: Isolated CR-CSCs have a normal phenotype, and can be spherical growth. In addition, the CR-CSCs can express pluripotency gene Sox2, Oct4 and Nanog. Heat shock has no influence on the morphology and surface marker of EXO(HS-EXO). Under the HS-EXO stimulation, PBMCs proliferation and killing capacity has been significantly improved(P<0.05), and can significantly promote IFN-γ and IL-2 secretion, and also improve the expression of immune related molecules HSP60, HSP90, HLA-1 and ICAM-1. In vivo results showed: Isolated CR-CSCs have strong tumorigenic ability, all mice of PBS control group have grown tumor after inoculation 13 d. The time that mice begin to grow tumor is significantly delayed after HS-EXO and EXO immunization, the number of tumor free mice is significantly lower than PBS control group(P<0.001). The preventive effect of HS-EXO is a little better than EXO, but there is no significant difference between the two groups. After HS-EXO and EXO immunotherapy, both groups significantly reduce the tumor growth rate, and the number of tumor free mice and survival rate are significantly higher than control group. The therapeutic effect of HS-EXO is significantly better than EXO, there is a significant difference in the number of tumor free mice.In summary, EXO are used as the core of this project and CR-CSCs are used as the basis of this project to explore the impact of heat shock on the immunological properties of CR-CSCs-derived EXO. We found that heat shock does not affect the basic nature of EXO, and can also increase the expression of HSP and immune-related proteins of EXO. Therefore, heat shock can improve the immunogenicity of EXO,and then improve the immune preventive function and immune therapeutic function.This will provide certain scientific data for the clinical application of EXO vaccine.