The Mechanism Of Regulating Dendritic Cell Function By Changed Tumor Microenvironment In Colorectal Cancer Cell Without Ese-3 Expression

BackgroundColorectal cancer is one of the most common malignant tumors of the digestive tract in the world.With the change of Chinese people's diet habits,the prevalence of colorectal cancer in China has increased year by year,and age of diagnosis.Colorectal cancer is difficult to be found by patients themselves.The clinical diagnosis of the disease often has been developed to advanced colorectal cancer.In the usual,many patients who have undergone multiple cycles of radiotherapy and chemotherapy after surgery have often experienced clinical drug resistance.It will lead to no medical treatment that can effectively control recurrence and metastasis of colorectal cancer,and the end result of this circumstance is the death of the patient.In recent years,with the rapid development of tumor immunotherapy and immunological research,immune cells and tumor microenvironment gradually become the focus of tumor research.However,the effect of immunotherapy for colorectal cancer is not satisfactory.It is very important to explore the mechanism of immunotherapy for colon cancer to explore the mechanism of colorectal cancer immunotherapy is very important.Dendritic cells,as an important antigen-presenting cell,play a very important role in tumor immunity.Ese-3 as a transcription factor,by regulating the expression of a variety of genes,affect cell proliferation,apoptosis and transformation,in the tumor is closely related.Previous studies have shown that the expression of the molecule in colon cancer is down-regulated and is closely related to poor prognosis in colon cancer patients.However,little attention has been paid to the expression of Ese-3 gene in colon cancer cells and microenvironment and immune cells.ObjectiveWe constructed SW480 Ese-3,a colorectal cancer cell line that overexpressed Ese-3,in order to co-culture with DC cells.Then,we analyze the ability of DC cells to secrete cytokine IL12-p70,the expression of DC cell surface molecular markers,phagocytic capacity of DC cells,and the ability of DC cells to activate T cells.Through the above results,we tried to find out whether the expression level of Ese-3 in colorectal cancer cells can influence the microenvironment and the differentiation and maturation of DC cells.Methodslentivirus-mediated biological transfection technique was used to build a stable expression of Ese-3 gene in SW480 cell line(SW480 Ese-3)and its relative controls(SW480 NC).CD14+ cells were obtained from human peripheral blood mononuclear cells(PBMC)by magnetic bead sorting and induced to produce iDC by IL-4 and rhGM-CSF.The iDC were stimulated by TNF-?,and co-cultured with SW480 Ese-3 and its control cell line SW480 NC respectively by Transwell chamber.The expression of HLA-DR,CD14,CD83 and CD86 in co-cultured DC cells was detected by flow cytometry.The level of IL-12p70 in supernatant of cell culture was detected by ELISA.The co-cultured DC cells were collected and cultured with FITC-Dextran.Flow cytometry was used to detect the phagocytosis of DC cells.After the co-culture of DC cells with T cells,MTT assay was used to detect the proliferation of T cells ability.Results(1)SW480 Ese-3 cells and their control cells SW480 NC were obtained after lentivirus infection.Western blot analysis showed that Ese-3 expression in SW480 Ese-3 cells was higher than SW480 NC cells.Obtained stable expression of Ese-3 human colon cancer cell SW480 Ese-3.(2)CD14 + cells were obtained from PBMC by magnetic bead sorting technique and DC cells were induced in vitro.The untransfection system of colon cancer cells and DC cells was successfully constructed by Transwell chamber.(3)SW480 Ese-3 cells and their control cells were co-cultured with DC cells respectively.The concentration of IL-12p70 in culture supernatant was detected by ELISA.The levels of IL-12p70 in SW480 Ese-3/DC co-culture group were high The SW480 Ese-3 cells were able to promote the secretion of IL12-p70 by DC cells.(4)DC cells were detected by flow cytometry.The levels of HLA-DR,CD14,CD83 and CD86 on DC cell membrane were detected by flow cytometry.The expression of CD83 and CD86 on SW480 Ese-3/DC co-culture group Compared with SW480 NC/DC co-culture group,CD14 level decreased,suggesting that SW480 Ese-3 cells can promote the expression of CD83 and CD86 in DC membrane and inhibit the expression of CD14.(5)The co-cultured DC cells were collected and cultured with FITC-Dextran.Flow cytometry was used to detect the phagocytosis of DC cells.The results showed that Dextran in SW480 Ese-3/DC co-culture group was lower than that in control group SW480 Ese-3/DC co-culture group DC phagocytosis was weaker than the control group.(6)The co-cultured DC cells were collected for mixed lymphocyte reaction.MTT results: SW480 Ese-3/DC co-culture group had stronger ability to promote T cell proliferation compared with DC cells in control group,And the ratio of DC cells and T cells is 1: 5,this effect is most obvious.ConclusionThe results of the above study,with the co-culture system,confirmed that the expression of Ese-3 in colorectal cancer cells can promote the differentiation and maturation of DC.This result suggests that Ese-3 can regulate the microenvironment of colorectal cancer cells and thus influence the differentiation and maturation of DC cells in co-culture system,which may play a role in the process of colorectal cancer immune escape.This study laid a foundation for further exploring the mechanism of differential expression of Ese-3 in colorectal cancer cells to differentiate and maturation of DC cells and provide a new research direction for the development and immunotherapy of colorectal cancer.