Study Of The Tracer Of EGFR-targeted CAR-T Cells And The Killing Effection In Breast Cancer Cell Line

Due to the poor prognosis of triple-negative breast cancer(TNBC),a new treatment is urgently needed to improve its efficacy.Chimeric antigen receptor(CAR)T-cell therapy has shown a great clinical effection in hematologic malignancies,and has also been gradually extended to the preclinical study of solid tumors.However,the work way of CAR-T cells has not been clearly understand,the application of tracer technology can better understand the biological characteristics between these cells while also play an important role in optimizing CAR-T cell immunotherapy.Epidermal growth factor receptor(EGFR)is highly expressed in 45-70% of TNBC,suggesting that it’s feasible to be a target of TNBC.Therefore,this study intended to construct three third-generation EGFR-CAR Jurkat cells carrying RFP and EGFR-positive breast cancer cells carrying EGFP,which were both constructed by lentivirus transfection and purified by selective monoclonal method.The proliferation of four Jurkat cells was detected by CCK-8 assay.The expression of EGFR-CAR-RFP gene was analyzed by Real-Time PCR and Western-blot.The secretion of IL-2 in cultured supernatant was detected by ELISA.Firstly,by cloning the third generation CAR gene,three lentiviral vectors carried RFP and three recombinant CAR-T lentivirus were successfully constructed.Then,three T cell lines with stable transfection of EGFR-CAR-RFP gene and one EGFR-positive breast cancer cell line with stable transfection of EGFP gene by means of cloning selection successfully were successfully obtained.Finally,Real-Time PCR results showed that exogenous CD3ζ m RNA level was significantly increased in all three Jurkat cells transfected with the EGFR-CAR-RFP gene compared with non-transfected Jurkat cells(P<0.001).Western-blot results also showed that exogenous CD3ζ protein level in all three CAR Jurkat cells transfected with the EGFRCAR-RFP gene was higher than non-transfected Jurkat cells.CCK-8 assay showed that there was no statistical difference in proliferation between Jurkat cells transfected with EGFR-CAR-RFP gene and non-transfected Jurkat cells(P>0.05).ELISA results showed that the secretion of IL-2 in E:T= 1:1,the two groups co-cultured with EGFR-CAR-RFP Jurkat cells was significantly higher than which co-cultured with Con-CAR-RFP Jurkat cells(P<0.001).With the increase of co-culture proportion,compared with the co-culture group of Con-CAR-RFP Jurkat cells,the secretion of IL-2 in the co-culture group of EGFR-CAR1-RFP Jurkat cells was significantly increased(P<0.01)as well as the co-culture group of EGFR-CAR2-RFP Jurkat cells(P<0.001).In summary,three EGFR-targeted CAR Jurkat cells were successfully constructed in this study,and the specific recognition of EGFR-positive breast cancer cells MDA-MB-468 was verified,which provides experimental reference for the application of EGFR-CAR modified PBMC-derived T lymphocytes in the cell immunotherapy of triple-negative breast cancer.