通过构建胃癌类器官共培养模型探究嵌合抗原受体T细胞治疗与临床病理的关系  

Gastric organoid co-culture model for investigating the relationship between chimeric antigen receptor T cells therapy and clinicopathological features Fang

作　　者：方伟超 王晨[1,2] 刘馨然 李宽 侯浩斌 陈伟 李明哲[1,2] 何裕隆 张常华[1,2] Weichao;Wang Chen;Liu Xinran;Li Kuan;Hou Haobin;Chen Wei;Li Mingzhe;He Yulong;Zhang Changhua(Digestive Diseases Center,the Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen 518107,China;Guangdong Provincial Key Laboratory of Digestive Cancer Research,Shenzhen 518107,China)

机构地区：[1]中山大学附属第七医院消化医学中心,深圳518107 [2]广东省消化系统恶性肿瘤防治研究重点实验室,深圳518107

出　　处：《中华普通外科学文献(电子版)》2025年第2期89-95,共7页Chinese Archives of General Surgery(Electronic Edition)

基　　金：广东省消化系统恶性肿瘤防治研究重点实验室开放基金项目(2021B1212040006);深圳市“医疗卫生三名工程”资助项目(SZSM201911010);深圳市医学重点学科建设基金项目(SZXK016);深圳市可持续发展科技专项(KCXFZ20200201101059392);深圳市科技创新委员会科技重大专项(KJZD20230923114101003)。

摘　　要：目的构建胃癌类器官与CLDN18.2-嵌合抗原受体T细胞(CAR-T)共培养模型,探究CAR-T杀伤效率与临床病理因素,尤其是CLDN18.2表达强度的关系。方法收集2022年9月至2023年8月中山大学附属第七医院收治的73例胃癌患者临床病理资料。采用免疫组织化学染色法检测肿瘤组织CLDN18.2表达。构建胃癌类器官并通过免疫组织化学和免疫荧光染色验证CLDN18.2表达特征。构建CLDN18.2-CAR-T细胞,将其与12例胃癌类器官共培养,采用乳酸脱氢酶(LDH)释放实验、酶联免疫吸附测定(ELISA)细胞因子检测及荧光杀伤成像评估CAR-T杀伤效率。单因素分析和相关性分析探究CLDN18.2表达强度与CAR-T杀伤效率的关系。结果体外培养类器官保留了原发肿瘤的CLDN18.2表达特征。共培养实验证明CAR-T能够有效杀伤类器官。临床病理单因素分析证实,CLDN18.2表达强度是影响CAR-T杀伤效率的因素。相关性分析提示,杀伤效率与CLDN18.2表达水平呈显著正相关(r=0.73,P=0.007)。结论成功构建了用于体外验证CAR-T治疗效果的共培养模型,并成功制备出具有显著活性的靶向CLDN18.2的CAR-T。Objective To construct a co-culture model of gastric cancer organoids and CLDN18.2-chimeric antigen receptor T(CAR-T)cells,and to investigate the relationship between CAR-T cytotoxicity and clinicopathological factors,especially the intensity of CLDN18.2 expression.Methods Clinical and pathological data were collected from 73 patients with gastric cancer admitted in the Seventh Affiliated Hospital of Sun Yat-sen University from September 2022 to August 2023.CLDN18.2 expression in tumor tissues was detected using immunohistochemical staining.Gastric cancer organoids were established and the CLDN18.2 expression profile was verified by both immunohistochemical and immunofluorescence staining.CLDN18.2-CAR-T cells were developed and co-cultured with 12 gastric cancer organoid models.CAR-T cell cytotoxicity was evaluated using lactic dehydrogenase(LDH)release assays,enzyme linked immunosorbent assay(ELISA)for cytokine detection,and fluorescence imaging of cell killing.Univariate and correlation analyses were performed to explore the relationship between CLDN18.2 expression and CAR-T cell cytotoxicity.Results Organoids retained the expression characteristics of CLDN18.2 in the primary tumors.Co-culture experiments demonstrated that CAR-T cells killed the organoids effectively.Univariate analysis of clinicopathological factors confirmed that CLDN18.2 expression intensity was a factor influencing CAR-T cell cytotoxicity.Correlation analysis indicated significantly positive correlation between cytotoxicity and CLDN18.2 expression level(r=0.73,P=0.007).Conclusion A successful co-culture model was constructed for in vitro validation of CAR-T therapeutic efficacy and development of highly active CLDN18.2-targeted CAR-T cells.

关 键 词：胃肿瘤 类器官 CLDN18.2 CAR-T 嵌合抗原受体 

分 类 号：R735.2[医药卫生—肿瘤]