Shi Hui, Yu Feng, Mao Yinting, Ju Qianqian, Wu Yingcheng, Bai Wen, Wang Peiwen, Xu Ran, Jiang Maorong, Shi Jiahai
Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China.
Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
J Thorac Dis. 2018 May;10(5):2779-2788. doi: 10.21037/jtd.2018.04.91.
It is urgent to explore an effective potential therapeutic strategy for ESCC. In recent years, cell-based cancer immunotherapy has become a potentially close for carcinoma therapy. Chimeric antigen receptor (CAR) T cell technology is a kind of adoptive cell therapy technique which has been developed rapidly. We sought to obtain EphA2.CAR-T cell and revealed the ability of EphA2.CAR-T cells to kill esophageal squamous cell carcinoma (ESCC) cells .
Firstly, the expression and location of EphA2 in ESCC tissues and cells was tested by immunohistochemistry staining and Western blot. Secondly, the second generation of EphA2.CAR was constructed via molecular biology technology, and transduced into T cells to obtain the EphA2.CAR-T cell. The transduction efficacies were assessed using flow cytometry (FCM). Thirdly, the effect of cell killing of EphA2.CAR-T cell on ESCC cells was detected by co-culture experiments. The productions of cytokines (TNF-α and IFN-γ) by EphA2.CAR-T cell after co-culture with ESCC cells were analyzed by ELISA assay.
The expression of EphA2 was significantly upregulated in ESCC tissues and cells (P<0.05). EphA2 was expressed on the membrane of ESCC cells, so it could be served as tumor-associated surface antigens (TAA) of CAR for ESCC treatment. The EphA2.CAR-T cell was obtained successfully, and its' transduction efficacies was 61.4% by FCM. The ability of cell killing of EphA2.CAR-T cell was better than that of T cells (P<0.01), and demonstrated a dose-dependent cell killing. The results of ELISA assay showed that the levels of TNF-α and IFN-γ in EphA2.CAR-T cells were notably raised compared with T cells (P<0.05).
We firstly constructed the second generation of EphA2.CAR and established EphA2.CAR-T cells. The EphA2.CAR-T cells showed a dose-dependent cell killing of ESCC cells, and promoted the production of cytokines . These findings open a new way for treatment of ESCC by immunotherapy in the future.
探索一种有效的食管癌潜在治疗策略迫在眉睫。近年来，基于细胞的癌症免疫疗法已成为一种极具潜力的癌症治疗方法。嵌合抗原受体（CAR）T细胞技术是一种发展迅速的过继性细胞治疗技术。我们试图获得EphA2.CAR-T细胞，并揭示EphA2.CAR-T细胞杀伤食管鳞状细胞癌（ESCC）细胞的能力。
首先，通过免疫组织化学染色和蛋白质免疫印迹法检测EphA2在ESCC组织和细胞中的表达及定位。其次，通过分子生物学技术构建第二代EphA2.CAR，并转导至T细胞中以获得EphA2.CAR-T细胞。使用流式细胞术（FCM）评估转导效率。第三，通过共培养实验检测EphA2.CAR-T细胞对ESCC细胞的杀伤作用。通过酶联免疫吸附测定（ELISA）分析EphA2.CAR-T细胞与ESCC细胞共培养后细胞因子（TNF-α和IFN-γ）的产生情况。
EphA2在ESCC组织和细胞中的表达显著上调（P<0.05）。EphA2表达于ESCC细胞膜上，因此可作为用于ESCC治疗的CAR的肿瘤相关表面抗原（TAA）。成功获得了EphA2.CAR-T细胞，通过FCM检测其转导效率为61.4%。EphA2.CAR-T细胞的杀伤能力优于T细胞（P<0.01），并呈现出剂量依赖性的细胞杀伤作用。ELISA检测结果显示，与T细胞相比，EphA2.CAR-T细胞中TNF-α和IFN-γ的水平显著升高（P<0.05）。
我们首次构建了第二代EphA2.CAR并建立了EphA2.CAR-T细胞。EphA2.CAR-T细胞对ESCC细胞呈现出剂量依赖性的细胞杀伤作用，并促进了细胞因子的产生。这些发现为未来通过免疫疗法治疗ESCC开辟了一条新途径。
