结直肠癌类器官的反应性T细胞受体筛选及功能鉴定  

Screening and identification of tumor-specific T cell receptors from tumor infiltrating lymphocytes using colorectal cancer organoid model

作　　者：梅瑶瑶 王应明 韩晓建 申美莹 李娅 魏正强[4] 金艾顺 MEI Yaoyao;WANG Yingming;HAN Xiaojian;SHEN Meiying;LI Ya;WEI Zhengqiang;JIN Aishun(Department of Immunology,School of Basic Medical Sciences,Chongqing Medical University,Chongqing 400016,China;Chongqing Key Laboratory of Tumor Immune Regulation and Immune Intervention,Chongqing 400016,China;Department of Breast and Thyroid Surgery,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Gastrointestinal Surgery,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院免疫学教研室,400016 [2]重庆医科大学肿瘤免疫调节与免疫干预重庆市重点实验室,400016 [3]重庆医科大学附属第一医院乳腺外科,400016 [4]重庆医科大学附属第一医院胃肠外科,400016

出　　处：《免疫学杂志》2024年第3期279-285,共7页Immunological Journal

基　　金：重庆市自然科学基金博士后科学基金项目(cstc 2020jcyi-bshX0020)。

摘　　要：目的本研究通过建立基于结直肠癌(colorectal cancer,CRC)类器官模型以及CRC反应性肿瘤浸润淋巴细胞(tumor-infiltrating lymphocyte,TIL)扩增和分离,筛选肿瘤特异性T细胞受体(T cell receptor,TCR)并进行功能验证,为结直肠癌个体化过继性T细胞免疫治疗的临床转化提供技术平台和研究基础。方法通过体外3D培养技术构建结肠癌患者组织来源的类器官模型,并利用HE染色和免疫组化进行形态学和特征分子表达检测。随后,将CRC类器官与TIL共培养,通过流式细胞技术分选反应性TIL,通过单个T细胞受体基因克隆技术分析反应性TCR克隆特征。进一步通过细胞毒性实验对TCR功能进行验证。结果通过形态学、代表性分子(CK20和CDX2)表达水平验证了结直肠癌类器官与患者肿瘤具有高度相似性。成功筛选到CD137表达上调和IFN-γ分泌增加的结直肠癌反应性T细胞,其中TCR2-T展现出较好的肿瘤反应性和体外肿瘤杀伤功能。结论建立了基于CRC-Org的反应性TCR筛选和功能验证平台,为结直肠癌个体化T细胞治疗转化应用提供技术平台。This study intends to establish a colorectal cancer(CRC)organoid model,expand and isolate CRC-reactive tumor-infiltrating lymphocytes(TILs),screen tumor-specific T cell receptors(TCRs)and perform functional verification,in order to provide a technological platform and research foundation for the clinical transformation of individualized adoptive T-cell immunotherapy for colorectal cancer.An organoid model derived from colon cancer patient tissues was constructed using in vitro 3D culture techniques,which then subjected to HE staining and immunohistochemistry for detecting morphological characteristics and representative molecular expression.Subsequently,CRC organoids were co-cultured with TILs for sorting reactive TILs using flow cytometry,and the characteristics of reactive TCR clones was analyzed through single T cell receptor gene cloning technology.Furthermore,the function of TCRs was verified through cytotoxicity experiments.Morphological analysis and representative molecules(CK20 and CDX2)expression indicated that there is high similarity between colorectal cancer organoids and patient tumors.In the in vitro expanded and cultured TILs,colorectal cancer-reactive T cells with upregulated CD137 expression and increased IFN-γsecretion were screened out successfully,among which TCR2-T cells demonstrated superior tumor reactivity and in vitro tumor killing function.In conclusion,a platform for screening and function validation of reactive TCRs based on CRC-Org has been established,providing a technological platform for the translational application of individualized T-cell therapy for colorectal cancer.

关 键 词：结直肠癌类器官 肿瘤反应性T细胞 T细胞受体 抗肿瘤 

分 类 号：R735.3[医药卫生—肿瘤] R392[医药卫生—临床医学]