Background and objectivesIn China,esophageal cancer(EC)has lofty morbidity and mortality among all the tumors.ESCC is the predominant pathohistological type and accounts for 90%of EC.Particularly,ESCC has a very high incidence in Henan Province.Unfortunately,the 5-year survival of EC is around 20%or lower,although clinical regimens including surgery,chemotherapy and radiotherapy have improved the prognosis of EC patients.It’s with prior urgency to develop more effective therapy for patients acquiring EC.CAR-T cells have attained great success in battle with hematologic malignancies.However,the effectiveness of CAR-T cell therapy is not satisfying in solid tumors,which are heterogeneous in expression of antigen.In clinical trials,CAR-T cells that target a wide range of highly expressed antigens have achieved good results and even induced tumor regression in solid tumors.These results suggest that the key for optimal CAR-T cell therapy in solid tumors is finding out antigens that have extensive and high expression in malignant lesions.Currently,limited efforts have been made to explore CAR-T cell therapy in EC and it is unclear if there are antigens fitting for EC therapy.CD276,also known as B7H3,is an immune checkpoint member of the B7 superfamily.It has been reported that CD276 is highly expressed in a range of solid tumors but has low levels of expression in normal tissues and marginal tissues.CD276 overexpression is associated with tumor progression,metastasis,proliferation and poor clinical prognosis.Meanwhile,it has been confirmed that CD276 is highly expressed in EC,but it is still unclear whether CD276 is conformably overexpressed in EC tumor tissues.This project aims to explore CD276 expression in ESCC tissues and its impacts on proliferation of ESCC cells,then develop CD276-specific CAR-T cells and test the anti-tumor effects of CAR-T cells in ESCC in vitro and in vivo.Therefore,this project may develop novel CAR-T cell therapy fitting for ESCC,to provide a more effective strategy for the clinical treatment of ESCC and improve the prognosis and survival of ESCC patients.Methods1.Expression of CD276 in paraffin sections of tumor tissues and marginal tissues from ESCC patients and normal tissues from health subjects was detected by IHC.2.Expression of CD276 was detected in ESCC cell lines by FACS.3.The correlation between CD276 expression in tumor tissues of ESCC patients and malignancy grade was analyzed;and the online database was utilized to analyse the correlation between CD276 expression and tumor cell proliferation indicator Ki67 was analyzed.4.After CD276 knockdown efficiency by RNAi was examined by qPCR and WB,apoptosis and Ki67 expression of tumor cells were detected by FACS and tumor cell proliferation was assessed by BLI.5.CD276 expression in CD276 knockout cells by CRISPR/cas9 technology was examined by FACS and WB,and tumor cell proliferation was assessed by FACS and BLI.6.CAR-T cells were generated by magnetic cell sorting and lentivirus transduction,and the cytotoxic functions of CAR-T cells were analyzed by FACS and ELISA.7.The effects of soluble CD276 on CD276-specific CAR-T cells were monitored by BLI and ELISA.8.NSG mice with established xenograft tumors were treated with CAR-T cells via tail vein injection.Then,the ratios and activities of intratumoural CAR-T cells were checked by FACS,tumor growth was measured by BLI and survival time of mice was recorded.9.CAR-T cells were generated from T cells of ESCC patients,and efficacy of CAR-T cells killing primary tumor cells was evaluated by FACS.10.NSG mice bearing PDX models were injected intravenously with CAR-T cells via the tail vein,then tumor growth and survival of mice were monitored.Results1.CD276 had low levels of expression in limited cells in normal tissues,whereas CD276 was conformably overexpressed in ESCC tumor tissues.2.CD276 overexpression indicated greater tumor sizes,elevated lymph node invasion,poorer differentiation and advanced stages.3.Knockdown or knockout of CD276 largely inhibit proliferation in ESCC cells.4.CD276-specific CAR-T cells were constructed successfully.5.Soluble CD276 had no effect on antitumor activity of CD276-specific CAR-T cells.6.CD276-specific CAR-T cells efficiently delayed tumor growth and improved survival in xenograft tumor models.7.CD276-specific CAR-T cells efficiently killed primary tumor cells and tissues.ConclusionCD276 was widely overexpressed in ESCC tumor tissues but had low levels of expression in normal tissues,and CD276 had tumor-promoting roles.Those results suggested that CD276 is a good target for CAR-T cell therapy in ESCC.CD276-specific CAR-T cells effectively killed primary tumor cells and tissues.CD276-specific CAR-T cells delayed tumor growth and improved survival.Therefore,CD276-specific CAR-T cells is probably an effective therapy for patients with ESCC. |