Effect Of C-Met Targeting CAR-T Cells On Human Colorectal Cancer Cells

Background:Colorectal cancer is one of the malignant tumors threatening human life,and its incidence is increasing gradually.Most patients still use traditional chemotherapy,radiotherapy or surgery as the main treatment,but have not achieved satisfactory therapeutic effect.In recent years,Chimeric antigen receptor T cell(CAR-T)therapy has made great breakthroughs in the treatment of hematological malignancies such as B cells,but there has been no significant progress in the treatment of solid tumors.cellular mesenchymal epithelial transition factor(c-Met)was highly expressed on the surface of various solid tumors such as gastric cancer,liver cancer and colorectal cancer,but was expressed at low level or not on the surface of normal tissue cells.CAR-T cells targeting c-Met have shown great potential in the treatment of solid tumors.Objective:Based on bioinformatics and immunohistochemistry of clinicopathological specimens,the expression of MET in colorectal adenocarcinoma and its influence on the progression and prognosis of colorectal adenocarcinoma were analyzed.We prepared CAR-T cells targeting c-Met and observed its killing effect on colorectal cancer cell HCT116 in vitro.Methods:1.The expression of MET gene in colorectal adenocarcinoma was preliminarily discussed through GEPIA and HPA databases.Meanwhile,GEPIA was used to analyze the expression difference of MET in pathological stagesⅠ,Ⅱ,ⅢandⅣof colorectal adenocarcinoma and the effect on survival and prognosis of patients.The UALCAN database was used to analyze the relationship between MET gene expression and histological subtypes of colorectal adenocarcinoma,as well as patient race,sex,weight,age,and lymph node metastasis status.The relationship between MET gene and the abundance of immune cell infiltration was analyzed by TIMER database.Cancer SEA database analyses the relationship between MET gene expression and angiogenesis,cell cycle,diffusion,stem cell characterization,inflammation,metastasis,and epithelial interstitial transformation.MET mutations in colorectal adenocarcinoma were analyzed by c Bio Portal database.Protein interaction network map was made with STRING database to analyze the effects of MET and interactive proteins.Immunohistochemical staining was performed on 40 colorectal cancer tumor tissues and 20 paracancer tissues from the Department of Pathology,the First Affiliated Hospital of Bengbu Medical College to verify the expression of c-Met in colorectal cancer.2.The second generation of c-Met CAR-T cells and CD19 CAR-T cells were prepared by lentivirus infection,and the expressions of CAR sequences were verified by Western blot.The positive rate of c-Met CAR-T cells and CD19 CAR-T cells was determined by flow cytometry,and the subpopulation ratio of T,c-Met CAR-T cells and CD19 CAR-T cells was determined by flow cytometry.Group the in vitro experiments.Experimental group:c-Met CAR-T cells+HCT116 cells.Control group:CD19 CAR-T cells+HCT116 cells,T cells+HCT116 cells.Non target group:c-Met CAR-T cells+A2780 cells,CD19 CAR-T cells+A2780 cells,T cells+A2780 cells.The expression of c-Met protein on the surface of colorectal cancer cancer cell HCT116and ovarian cancer cell A2780 was verified by flow cytometry.CCK-8 was used to detect the proliferation of c-Met CAR-T cells at 24h and 48h.LDH was used to determine the killing rate of c-Met CAR-T cells against HCT116 cells at the effect-target ratio of 1:1,5:1,10:1 and 20:1.ELISA was used to detect the secretion of cytokines IL-2 and IFN-γin c-Met CAR-T stimulated by target antigen.Results:1.GEPIA and HPA databases showed that MET gene expression was significantly higher in colorectal adenocarcinoma(P<0.01).GEPIA also found that MET gene expression was not correlated with pathological staging,overall survival and disease-free survival.Immunohistochemistry further confirmed that c-Met was highly expressed in colon cancer tissues,with a strong positive expression rate of 77.5%.The UALCAN database showed that MET expression was associated with histological subtypes of colorectal adenocarcinoma(P<0.001)and was independent of patient race,sex,weight,age,and lymph node metastasis.The TIMER database showed that the expression of MET gene was correlated with the infiltration level of immune cells to colorectal adenocarcinoma(P<0.01).The mutation rate of MET gene in colorectal adenocarcinoma was 3.30%in c Bio Portal database.The Cancer SEA database shows that MET gene expression is positively associated with angiogenesis,cell cycle,diffusion,stem cell characterization,inflammation,metastasis,and epithelial mesenchymal transformation in colorectal adenocarcinoma(P<0.05).In the STRING database,HGF was found to have a higher protein score interacting with MET and was in the first place.2.Western blot results showed that CAR molecules existed in CAR-T cells after lentivirus infection,indicating successful construction of second-generation c-Met CAR-T cells.The positive rates of c-Met CAR-T cells and CD19 CAR-T cells were42.9%and 91.9%,respectively,by flow cytometry.For c-Met CAR-T cells,the percentage of CD8~+T cells was up-regulated after lentiviral infection.Flow cytometry showed high expression of c-Met on HCT116 and negative expression of c-Met on A2780.CCK-8 results showed that the proliferation potential of c-Met CAR-T cells was significantly enhanced in the presence of target antigen.LDH release assay showed that the anti-tumor effect of c-Met CAR-T cells increased with the increase of efficacy target ratio.When the effect target ratio was 20:1,the killing rate reached the highest(64.18%).ELISA results showed that,compared with CD19 CAR-T cells and activated T cells,c-Met CAR-T cells could release more IL-2 and IFN-γwhen stimulated by target cells.Conclusions:1.c-Met can be used as a molecular target of CAR-T cell therapy for colorectal cancer.2.c-Met CAR-T cells can target to recognize and kill c-Met positive colorectal cancer cells,while improving proliferation and cytokine release levels.