肝癌类器官模型的建立及个性化药物筛选和疗效评估的研究

As a new type of tumor research model, organoid can stably maintain the characteristics of tumor cells in vivo. At present, it has been used as a drug screening and therapeutic evaluation model and applied to a variety of tumor research. However, due to the low survival rate and stable passage of liver cancer organoid, the related research progress is slow. In the early stage, we cooperated with the research group of Gao Dong, a researcher from the Institute of Biochemistry of the Chinese Academy of Sciences, using the clinically resected liver cancer tissue as the source, and optimized and improved four culture systems for liver cancer organoid with different mutation types, including TP53 and β-catenin mutations and EGFR amplification. So far, we have successfully constructed 30 human liver cancer organoids. Based on the above work, firstly, we will study the biological characteristics of five successfully constructed liver cancer organoids from three levels: morphology, function, and Genomics. Secondly, select five different anti-tumor drugs to compare the differences in drug sensitivity between In vitro 3D culture and PDO mouse model, and initially establish a drug sensitivity screening platform for liver cancer organoid culture. Finally, we will select three clinical first-line drugs, integrate transcriptomics data and conduct individualized sequential treatment of liver cancer. This investigation could provide a theoretical basis for the construction of functional human liver cancer organoid, and provide new technologies and strategies for personalized drug screening and efficacy evaluation of liver cancer.

类器官(organoid)作为一种新型肿瘤研究模型,可以稳定维持肿瘤细胞在体内特性，是目前理想的药物筛选和疗效评价模型，已被用于多种肿瘤的相关研究。然而肝癌类器官由于成活率低、稳定传代难导致相关研究进展缓慢。我们前期与中科院生化所高栋研究员课题组合作，使用临床手术切除的肝癌组织,优化完善了4种分别针对TP53、β-catenin以及EGFR突变的肝癌类器官培养体系，成功构建了30例人肝癌类器官。本课题拟在此基础上，首先对5例成功构建的肝癌类器官从形态学、功能学、基因组学三个层面开展生物学特性研究；其次选取5种不同作用机理抗肿瘤药物，结合体外3D培养和PDO小鼠模型建立肝癌类器官培养药物敏感性筛选平台；最后选用3种临床一线药物基于体外筛选平台，整合转录组学数据开展肝癌个体化序贯治疗研究。本课题的顺利实施将为构建功能性人肝癌类器官提供理论基础，为肝癌个性化药物筛选和疗效评估提供新技术和新策略。

本课题使用单细胞转录组测序技术重点分析了前期已建立的7例患者源性肝胆系统肿瘤类器官（Hepatobiliary Tumor Organoid）肿瘤间和肿瘤内异质性，发现并评估了与细胞周期、缺氧和上皮分化状态有关的特征基因，初步确定了与肝癌耐药相关的细胞亚群特征及可能的信号通路。发现具有上皮-间质转化状态的HCC272具有广谱耐药性，同时存在PI3K-Akt和MAPK信号通路组成性激活，CD44阳性亚群介导了HCC272中耐药性的产生。低氧信号富集的代谢优势肿瘤类器官亚群可以上调CD44亚组中NEAT1的表达，并通过配体-受体相互作用介导依赖Jak-STAT通路的耐药性。此外，还发现在多个类器官中共有的代谢优势簇具有相似的特征基因（GAPDH、NDRG1、ALDOA和CA9）。多因素分析发现GAPDH和NDRG1的组合是一个独立的危险因素，可作为患者生存的预测指标。该研究丰富了我们对肝癌细胞异质性介导耐药分子机、理的认识，也为肝胆肿瘤治疗提供潜在靶点。