靶向FRα嵌合抗原受体T淋巴细胞杀伤结直肠癌细胞的研究

Study on FRα targeted chimeric antigen receptor T lymphocytes killing colorectal cancer cells

目的探究叶酸受体α抗原(FRα)在结直肠癌中的表达情况,通过构建靶向FRα抗原的嵌合抗原受体(CAR)T淋巴细胞(FRα-CAR T),评估使用FRα-CAR T治疗结直肠癌的可行性。方法应用生物信息学数据库(TIMER2.0)分析FRα抗原在结直肠癌组织中的表达情况及其与免疫细胞浸润的关系。使用慢病毒载体将编码靶向FRα抗原的二代CAR结构的基因转导入CD8+T淋巴细胞,制备FRα-CAR T,流式细胞术分析转染效率。体外培养CACO-2和HCT-116细胞,采用流式细胞术分析FRα在肿瘤细胞表面的表达情况,将FRα-CAR T和CD19-CAR T及空载体转导的T细胞(mock T)作为效应细胞,CACO-2和HCT-116做为靶细胞,细胞计数试剂盒(CCK8)检测不同效靶比(1∶1、5∶1、10∶1和20∶1)对靶细胞的杀伤率;采用流式细胞术和ELISA法分别检测CAR T细胞杀伤CACO-2和HCT-116后细胞中干扰素γ(IFN-γ)和白介素-2(IL-2)的分泌情况。结果生物信息学分析显示FRα抗原在结直肠癌组织中表达水平高于正常组织(P=0.001),且其表达水平与结直肠癌组织中的DC细胞(r=-0.403)、NK细胞(r=-0.499)浸润呈负相关。使用慢病毒载体转导T细胞后,FRα-CAR表达率可达(41.6±4.5)%。CCK8实验显示随着效靶比提高,FRα-CAR T对CACO-2细胞杀伤效果逐渐增大,当效靶比为20∶1时,FRα-CAR T组、CD19-CAR T组和mock T组细胞毒性分别为(93.0±2.6)%、(15.4±2.3)%和(14.7±2.4)%,F=1705.943,P<0.001。CACO-2细胞中FRα-CAR T组、CD19-CAR T组、mock T组IL-2分泌量分别为(3700.0±264.6)、(128.5±21.7)和(125.3±25.2)pg/mL,F=897.583,P<0.001;FRα-CAR T组、CD19-CAR T组、mock T组IFN-γ阳性细胞比例分别为(22.9±3.3)%、(13.2±2.5)%和(14.5±2.4)%,F=18.159,P<0.001。结论FRα是结直肠癌治疗的潜在靶点,靶向FRα的CAR T细胞能够高效、特异性杀伤表达FRα的结直肠癌细胞。

Objective To explore the expression of folate receptorα(FRα)in colorectal tumor tissues and evaluate the feasibility of FRαchimeric antigen receptor of antigen T lymphocytes(FRa-CAR T)in the treatment of colorectal cancer by constructing targeted FRa-CAR T.Methods Bioinformatics database(TIMER2.0)was applied to analyze the expression level of FRαantigen in colorectal tumor tissues and the correlation with immune cell infiltration.Lentiviral vectors were used to deliver the gene into CD8~+T lymphocytes,which encoded a second-generation CAR structure targeting FRαantigen to prepare FRα-CAR T cells.Flow cytometry was used to analyze transfection efficiency.CACO-2 and HCT-116 cells were cultivated in vitro and flow cytometry was used to analyze expression of FRαon the surface of tumor cells.FRα-CART,CD19-CAR T and Mock T cells were used as effector cells,CACO-2 and HCT-116 were used as target cells.CCK8 assay was performed to detect the cytotoxicity of effector cells at different effector:target ratios(1:1,5:1,10:1 and 20:1 respectively).Flow cytometry and ELISA were performed to detect the secretion of interferonγ(IFN-γ)and interleukin-2(IL-2)after the co-incubation of effector cells with either CACO-2or HCT-116.Results Bioinformatics analysis showed that the expression level of FRαantigens in colorectal cancer tissue was higher than that in normal tissue(P=0.001),and its expression level was negatively correlated with the infiltration of DC cells(r=-0.403)and NK cells(r=-0.499)in colorectal cancer tissue.After using lentiviral vectors to transduce T cells,the CAR expression rate of FRα-CAR was(41.6±4.5)%.The CCK8experiment showed that as the efficiency to target ratio increased,the killing effect of FRα-CAR T on CACO-2cells gradually increased,and when the target effect ratio was20∶1,the cytotoxicity of FRα-CAR T group,CD19-CAR T group,and mock T group was(93.0±2.6)%,(15.4±2.3)%,and(14.7±2.4)%,respectively,F=1705.943,P<0.001.The IL-2secretion levels in FRα-CAR T group,CD19-CAR T group,and mock T group in CACO-2cells were(3700.0±264.6),(128.5±21.7),and(125.3±25.2)pg/mL,respectively,F=897.583,P<0.001.The proportion of IFN-γpositive cells was(22.9±3.3)%,(13.2±2.5)%,and(14.5±2.4)%,respectively in FRα-CAR T group,CD19-CAR T group,mock T group,F=18.159,P<0.001.Conclusions This study suggests that FRαis a potential target for the treatment of colorectal cancer,and CAR T cells targeting FRαcan efficiently and specifically kill FRα-expressing colorectal cancer cells.