表达荧光素酶的胰腺癌细胞系的建立及其在免疫细胞杀伤效力检测中的应用

Construction of luciferase-expressing pancreatic cancer cell lines and evaluation of their application in detecting cytotoxicity efficacy of immune cells

目的构建表达报告基因荧光素酶和肿瘤相关抗原间皮素(mesothelin,MSLN)基因的胰腺癌细胞系,评估其作为靶细胞在评价免疫细胞的肿瘤杀伤效力中的应用。方法构建表达荧光素酶、MSLN基因的慢病毒载体,包装慢病毒,感染胰腺癌细胞系,抗生素筛选后,有限稀释法获得单细胞克隆,并验证目的基因的稳定表达。实时无标记细胞分析(real-time cell analysis,RTCA)和荧光素酶活性检测方法体外检测免疫细胞对肿瘤细胞的杀伤效力。将构建的细胞系接种B-NDG小鼠建立表达荧光素酶的胰腺癌肿瘤模型,利用活体成像技术检测荧光素酶表达水平,监测小鼠体内胰腺癌肿瘤生长情况。在胰腺癌小鼠模型中验证嵌合抗原受体T(chimeric antigen receptor T,CAR-T)细胞的肿瘤杀伤能力。结果本研究建立了稳定表达荧光素酶和MSLN基因的胰腺癌细胞系panc-1-luc、panc-1-luc-MSLN和capan-2-luc细胞。3种细胞的报告基因表达水平较高,与细胞数量呈正相关,panc-1-luc-MSLN细胞的MSLN阳性率达95.6%。体外肿瘤细胞杀伤试验结果表明MSLN-CAR-T细胞能特异性杀伤MSLN抗原阳性的panc-1-luc-MSLN细胞和capan-2-luc细胞,最小杀伤率分别为(70.00±18.19)%和(57.00±5.29)%,而对MSLN阴性的panc-1-luc细胞无杀伤作用。RTCA结果显示MSLN-CAR-T细胞对构建的3种胰腺癌细胞均有杀伤能力,最小杀伤率分别为(56.33±7.64)%、(93.00±2.65)%和(26.33±28.15)%;NK-92MI细胞对靶细胞的杀伤不受MSLN限制。免疫缺陷小鼠胰腺癌模型结果显示,体内杀伤效力与体外荧光素酶活性检测结果一致。体内外试验证明,检测靶细胞荧光素酶表达活性,可以反映免疫细胞对靶细胞的杀伤效力。结论本研究建立了稳定表达荧光素酶的多株胰腺癌单克隆细胞系,可用于体内外免疫治疗产品肿瘤杀伤效力的研究评价。

Objective To construct pancreatic cancer cell lines expressing luciferase and mesothelin(MSLN),and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods Lentiviral vectors expressing luciferase and MSLN genes were constructed,and pancreatic cancer cell lines were infected after lentivirus packaging.Single-cell clones were obtained by limited dilution following antibiotic screening,and the stable expression of the target genes were verified.These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis(RTCA)and luciferase activity.Besides,these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer,and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice.The cytotoxicity of chimeric antigen receptor T(CAR-T)cells was verified in these animal models.Results Three pancreatic cancer cell lines,panc-1-luc,panc-1-luc-MSLN and capan-2-luc,that could stably express luciferase and MSLN genes were successfully constructed.The expression of the reporter gene in these cells were high,and positively correlated with the number of cells.There were 95.6%of panc-1-luc-MSLN cells expressing MSLN.MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells,with the minimum killing rates of(70.00±18.19)%and(57.00±5.29)%,respectively.But they had no cytotoxicity to MSLN-negative panc-1-luc cells.RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines,and the minimum killing rates were(56.33±7.64)%,(93.00±2.65)%and(26.33±28.15)%,respectively.The killing of target cells by NK-92MI cells was not depended on MSLN expression.The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro.The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells.Conclusions This study establishes three pancreatic cancer cell lines stably expressing luciferase,which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.