| Cancer is a serious disease threating human health and survival.Globally,ovarian cancer is the eighth most common cancer in women and the seventh most common cause of cancer death,with five-year survival rates below 45%,230,000 women are diagnosed with ovarian cancer every year.At the time of diagnosis,more than 75% of the cases are at advanced stage.Current common treatments for ovarian cancer include surgery,radiation and chemotherapy.These therapies can’t effectively control tumor recurrence and progress,and cannot effectively prolong survival time and improve patients ’ life quality.Therefore,it is urgent for us to find a new treatment.In recent years,biotherapy has become one of the most important ways for thetreatment of cancer.Studies have shown that that most of the cancer cells can evade the killing by the immune system,limiting the anti-cancer effects of immunotherapy.Therefore,a novel CAR-T technology with better targeting,killing,value-added and persistence was developed.A large number of clinical trials have shown that this technology has made great success in improving the quality of life and prolonging the survival time of patients with hematologic malignancies.Therefore,the CAR-T technology has become a hot research area.However,the research in solid tumors is not sufficient,and fail to get the expected effect owing to many obstacles,including: the specific pathological features of solid tumors,the lack of suitable surface antigen of the tumor,the immune suppression microenvironment,the optimal combination of costimulatory molecules,design of Suicide gene,adverse reactions after transfusion and so on.In this study,we constructed and tested the killing effect of mesoCAR-T on several ovarian cancer cell lines in vitro:1、we developed the second-generation mesothelin targeting CAR-T(mesoCAR-T)cells with 4-1BB and CD3 ζ co-stimulatory modules using piggyBac transposon system.of the transfection efficiency of mesoCar-T in T cells was about 30%.Results showed that mesoCAR could be stably expressed in mesoCAR-T cells.Flow cytometry(FCM)analysis showed that the expression of activation markers was significantly increased in mesoCAR-T transfected T cells after stimulation with meso.2、By comparing the expression levels of mesothelin among a variety of tumor cell lines using WesternBlot and Flow cytometry(FCM)analysis,we identified five ovarian cancer cell lines with different expression levels of mesothelin.We measured the killing effects of mesoCAR-T cells on high mesothelin expression cell line and low expression of mesothelial cell line.Our results showed the cytotoxic effect of mesoCAR-T cells in mesothelin-high cell lineswas significantly stronger than in mesothelin-low cell lines,indicating that the constructed mesoCAR-T cells were specific in targeting mesothelin-expressing cells.3 、 We successfully constructed mesoCAR-T.By detecting the mesoCAR-T cell cytokine after antigenstimulation,we found that the secretion of IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ was significantly higher than that of the control T cells.By detecting the mesoCAR-T cell cytokines after co-culture of mesoCAR-T cells with two kinds of ovarian tumor cells,It was found that the secretion of IL-2,IL-4,IL-6,IL-10、TNF-α,IFN-γwere significantly higher in the high mesothelin expression cell line(HO8910)than that in the low expression of mesothelial cell line(SKOV3).In summary,We successfully constructed mesoCAR-T,and the results showed that the laboratory piggyBac transposon system electroporation can be good for integrating foreign genes into T cells.mesoCAR-T cells can target mesothelin and effectively kill ovarian cancer cells,and the secretion of cytokines have demonstrated good results.Our study thus provides a basis for further investigation in vivo and clinical trials,and the development of new treatments for ovarian cancer. |
PDF Full Text Request