Spatial single cell analysis of tumor microenvironment remodeling pattern in primary central nervous system lymphoma. Spatial single cell analysis of tumor microenvironment remodeling pattern in primary central nervous system lymphoma

To determine the overall tumor microenvironment (TME), characteristics, and transition mechanisms in primary central nervous system lymphoma (PCNSL), we performed spatial transcriptomics and matched the corresponding single-cell sequencing data of PCNSL patients. We found that tumor cells may achieve a “TME remodeling pattern” through an “immune pressure-sensing model”, in which they could choose to reshape the TME into a barrier environment or a cold environment according to the immune pressure. A key FKBP5+ tumor subgroup was found to be responsible for pushing tumors into the barrier environment, which provides a possible way to evaluate the stage of PCNSL. The specific mechanism of the TME remodeling pattern and the key molecules of the immune pressure-sensing model were identified through the spatial communication analysis. Finally, we discovered the spatial and temporal distributions and variation characteristics of immune checkpoint molecules and CAR-T target molecules in immunotherapy. These data clarified the TME remodeling pattern of PCNSL, provided a reference for its immunotherapy, and provided suggestions for the TME remodeling mechanism of other cancers. Overall design: Here, 14964 single-cell transcriptomes of PCNSL were combined with spatial transcriptome analyses of hot, IME, IMS, and cold TMEs. The TMEs were characterized. In brief, the main cell types in the TME of PCNSL patients were identified by single cell sequencing, and were mapped to the corresponding spatial locations in 4 TMEs. Through integrative analysis, we defined and annotated tumor cell subpopulations according to their spatial distribution and functional characteristics and found that various tumor cells work together to reshape the TME into a barrier or cold environment through an "immune pressure-sensing model", thus realizing the "TME remodeling pattern". A key FKBP5+ tumor subgroup was found to be closely associated with TME remodeling, which provides a possible method for assessing brain tumor staging. The spatial communication mode between tumor cells and immune cells in each TME was also identified, thus refining the specific mechanism of the TME remodeling pattern and identifying the key molecules of the immune pressure-sensing model. Corresponding treatment suggestions were proposed according to the spatial characteristics of each TME in current PCNSL immunotherapy. These results reveal the spatial heterogeneity of PCNSL, highlight the localization and status of cell types and potential intercellular signals in the TME, and provide resources for further research on TMEs and updating immunotherapy methods.

为明确原发性中枢神经系统淋巴瘤（primary central nervous system lymphoma, PCNSL）的整体肿瘤微环境（tumor microenvironment, TME）特征及其转化机制，本研究针对PCNSL患者开展空间转录组学（spatial transcriptomics）分析，并匹配了对应的单细胞测序（single-cell sequencing）数据。研究发现，肿瘤细胞可通过“免疫压力感知模型”实现“TME重塑模式”：即肿瘤细胞可根据免疫压力状态，将TME重塑为屏障型微环境或冷肿瘤微环境。本研究鉴定出一类关键的FKBP5阳性（FKBP5+）肿瘤亚群，其可推动肿瘤向屏障型微环境进展，该亚群为PCNSL的临床分期评估提供了潜在可行途径。通过空间通讯分析，本研究明确了TME重塑模式的具体机制以及免疫压力感知模型的关键分子。最后，本研究揭示了免疫治疗中免疫检查点分子（immune checkpoint molecules）与CAR-T靶标分子（CAR-T target molecules）的时空分布及变异特征。上述研究结果阐明了PCNSL的TME重塑模式，为其免疫治疗提供了参考依据，并为其他癌症的TME重塑机制研究提供了借鉴思路。

整体实验设计：本研究整合了14964个PCNSL单细胞转录组数据，并针对热肿瘤微环境、免疫细胞浸润富集型（IME）、免疫细胞浸润中等型（IMS）及冷肿瘤微环境开展空间转录组分析，以系统表征PCNSL的TME特征。简言之，本研究通过单细胞测序鉴定了PCNSL患者TME中的主要细胞类型，并将其映射至4种TME的对应空间位置。通过整合组学分析，本研究根据肿瘤细胞的空间分布与功能特征对其亚群进行了定义与注释，发现不同肿瘤亚群可通过“免疫压力感知模型”协同将TME重塑为屏障型或冷肿瘤微环境，从而实现“TME重塑模式”。本研究进一步证实FKBP5+肿瘤亚群与TME重塑密切相关，为脑肿瘤分期评估提供了潜在方法。此外，本研究明确了各TME中肿瘤细胞与免疫细胞的空间通讯模式，从而细化了TME重塑模式的具体机制，并鉴定出免疫压力感知模型的关键分子。结合当前PCNSL免疫治疗中各TME的空间特征，本研究提出了对应的治疗建议。本研究结果揭示了PCNSL的空间异质性，阐明了TME中细胞类型的定位与状态以及潜在的细胞间信号通路，为TME的后续研究及免疫治疗方法的优化提供了重要的公共数据资源。