基于单细胞转录组测序的结肠癌肝转移的免疫特征研究

Tumor initiation, progression, and evolution are shaped not only by the selection of malignant cells with genetic alterations, but also by the influences of the tumor microenvironment, especially the infiltration of various immune cells. The traditional bulk expression profiles are based on the aggregates of diverse cells within each tumor sample, thereby masking critical differences between cancer and immune cells or immune cells themselves. Single-cell RNA sequencing (scRNA-seq) could help address these challenges. Colon cancer is one of the most common malignancies in China, with liver metastasis as the leading cause of colon-cancer-related mortality. Colon cancer with mismatch repair deficiency have been shown previously to be sensitive to immune checkpoint blockade with antibodies to programmed death receptor -1 (PD-1). However, there is still a lack of basic understanding of the composition, lineage and functional states of tumor-infiltrating immune cells in primary and metastatic lesion, as well as the differences among them. In this study, we plan to apply scRNA-seq analysis to >100,000 single immune cells isolated from the peripheral blood, primary and metastasized tumours, and corresponding adjacent normal tissues of colon cancer patients. We will perform a series of in-depth analyses of immune cells, including unsupervised or semi-supervised clustering into functional categories, inferring the trajectory of their development and differentiation, and remodelling their expression network. Furthermore, we will characterize the distinction of immune features between tumor and normal tissues, and between primary and metastatic lesions. Based on somatic variations in DNA and RNA level with expression network of immune cells, we plan to model the interactive evolutionary process of colon cancer liver metastasis. Finally, we will use both wet experimental approaches and large-scale genomic data mining to validate the findings derived from our high-throughput sequencing. In conclusion, we aim to generate and analyze scRNA data for a large compendium of infiltrating immune cells, providing insights into novel and effective immunotherapy strategies for colon cancer liver metastasis.

实体肿瘤中细胞成分高度异质，肿瘤细胞和肿瘤微环境细胞，特别是免疫细胞，共同塑造了肿瘤发生、发展、转归的各种表征。结肠癌肝转移恶性程度很高，从肿瘤免疫的角度对其进行研究具有重要生物学意义和临床前景。在本课题中，我们计划分析超过100,000个结肠癌肝转移相关的各类免疫细胞的转录组数据，推断细胞类型和细胞分化发育路径，重构肿瘤转移过程中的免疫细胞表达网络。整合癌组织体细胞变异与免疫细胞表达网络，推断其相互适应关系。比较不同器官癌灶免疫细胞的动员情况和发育分化路径，分析不同癌灶体细胞变异的差异对免疫细胞类别、状态和分化的影响。最后通过分子或免疫学实验以及大数据信息分析检验所发现的分子标志物或网络调控基因的生物学功能和临床意义。我们希望通过本研究中全面理解肿瘤转移过程中的免疫学特征，探索肿瘤细胞和免疫细胞在时空发展中的相互选择过程中的关键调控因子，为结肠癌肝转移提供新的治疗靶点或分子标志物。

利用面向大规模人群队列和单细胞水平的高通量测序数据，本研究共收集17例结肠癌肝转移患者，使用10x以及Smart-seq2单细胞测序技术分别产出了323,444和5,004个细胞的高质量转录组数据。我们对单细胞转录组开展了包括免疫细胞类型判断、表达特征鉴定、组织富集偏好分析、发育过程推断以及潜在功能发掘等生物信息分析的同时，对T细胞的TCR序列进行了组装，并进一步分析T细胞在各组织间的迁移情况。此外，通过结合TCGA的数据，我们从中验证了我们在单细胞水平上的发现，也为后续发现肿瘤转移机制和免疫细胞在转移过程中的功能等提供了临床价值。另外在本课题经费的部分资助下，我们结合甲基化和转录组测序，系统性地刻画了结直肠癌患者体内肿瘤特异性CD8+T细胞的甲基化和转录组特征，并探索了肿瘤特异性CD8+T细胞形成过程中潜在的关键调控因子。此外，我们同样采用单细胞转录组测序技术对肝癌和结直肠癌免疫微环境做出系统性刻画，不但揭示了新的细胞功能亚型，而且成为结肠癌肝转移研究的重要参照。最后，我们还开发了基于单细胞转录组数据进行快速有监督细胞类型注释的新工具，显著提升了单细胞转录组测序数据分析速率。综上，我们的工作从多方面揭示了结直肠癌肝转移患者的单细胞转录组特征和表达调控关系，探索了肝转移的发生机理，为癌症治疗以及靶向用药提供理论基础和技术支持。