基于滚环扩增的肿瘤浸润CD8+T细胞免疫状态的可视化研究

Tumor immune microenvironment plays an important role in tumorigenesis, development and therapeutic response. Tumor infiltrating CD8+ T cells play crucial roles on tumor killing, and their immune status and infiltrating numbers are significantly associated with clinical outcomes and prognosis of patients. So far, it is still lacking effective methods to precisely evaluate the immune status of tumor infiltrating cells; therefore, there is an urgent need to develop novel strategies for in situ analysis of immune status of infiltrating CD8+ T cells in tumor tissues. For resolving the issues of the diversity of immune status of CD8+ T cells, the cell heterogeneity and the complexity of tumor microenvironment, this project intends to achieve direct visualization of the immune status of tumor infiltrating CD8+ T cells, including: (1) specific gene expression patterns are taking as the marker of immune status of CD8+ T cells, then the immune status of CD8+ T cells was directly visualized at single-cell level by in situ rolling ring amplification (RCA); (2) a fluorescence spectral-coding method was constructed to realize the high-throughput analysis of specific genes relating to the immune status, as well as to meet the analysis requirements for immune status diversity of CD8+ T cells; (3) based on in situ imaging of the immune status markers and the spectral-coding strategy, the immune status of CD8+ T cells in tumor tissue is directly visualized. Above studies might help to construct molecular imaging based functional classification system, as well as to reveal the mechanism of immune microenvironment for influencing tumor cells.

肿瘤免疫微环境在肿瘤发生发展和治疗过程中扮演重要角色。肿瘤浸润的CD8+T细胞对肿瘤杀伤发挥关键作用，其免疫状态及数量与治疗疗效和病人预后密切相关。然而目前缺乏评估肿瘤浸润T细胞免疫状态的有效手段，急需发展肿瘤组织CD8+T细胞免疫状态的原位分析方法。针对CD8+T细胞免疫状态多样性，细胞异质性以及肿瘤组织微环境复杂性等问题，本项目拟开展以下工作：（1）以特异性基因表达作为CD8+T细胞免疫状态的标志物，利用核酸滚环扩增技术，实现CD8+T细胞免疫状态的单细胞分析；（2）构建基于核酸序列模块组合的荧光编码方法，实现免疫状态特征核酸的高通量分析，以满足CD8+T细胞免疫状态多样性分析的需求；（3）基于荧光编码策略和核酸标志物的原位成像分析，实现肿瘤组织CD8+T细胞免疫状态的可视化分析。上述研究的开展，有助于构建基于分子影像的功能分型指标体系，从而揭示免疫微环境影响肿瘤发生发展的机理。

肿瘤浸润T细胞对肿瘤杀伤发挥关键作用，其免疫状态、数量以及浸润深度与肿瘤免疫治疗疗效和病人预后密切相关。针对肿瘤浸润T细胞免疫状态多样性，细胞异质性以及肿瘤组织微环境复杂性等问题，本项目开发了一种基于滚换扩增技术的原位单细胞荧光成像方法，实现了临床标本中肿瘤浸润T细胞免疫状态、数量和位置的原位单细胞分析。主要研究内容包括：1）以CD8a mRNA作为CD8+T细胞的特征基因，建立基于滚环扩增技术的单细胞原位成像方法，并验证该方法具有高选择性和特异性；2）基于荧光光谱组合编码和滚环扩增技术，实现不同类型免疫细胞（CCRF-CEM/CD8+T/NK细胞）的单细胞原位成像，为发展高通量成像技术奠定基础；3）以4-1BB、PD1和Tim-3 mRNA作为特征基因，利用滚环扩增技术，构建CD8+T细胞激活（active）、“预耗竭”（pre-exhaustion）和耗竭（exhaustion）状态的原位可视化分析方法；4）在T细胞体外激活/扩增过程中，利用该方法监测其免疫状态特征基因表达量的变化规律；5）将上述构建的原位单细胞荧光成像方法，用于实际临床肿瘤组织切片中，肿瘤浸润T细胞的免疫状态、数量和位置信息的可视化，为构建基于分子影像的免疫分型指标体系奠定基础。