小胶质/巨噬细胞参与构建的胶质瘤微环境动态演进过程和靶点分子的功能及可视化研究

The highly heterogeneous and suppressive immune microenvironment of malignant glioma results in limited effect of the single molecular-targeted treatment or immunotherapy of glioma. Studies have showed that glioma infiltrating microglia /macrophages (GIM) could interact with tumor cells, activate cancer pathways and regulate immune microenvironment. Our previous studies have also found that GIM could interact with glioma stem cells and thus lead to the immune escape of glioma stem cells. Besides, GIM can downregulate the immune microenvironment of glioma through B7-H4. Therefore, we hypothesize that through further GIM researches, we may find new targeted molecules and pathways of glioma cell crosstalk and discover a "targeted immunotherapy" approach that may simultaneously suppress cancer pathways and reverse the immune microenvironment. This project plans to build a database of dynamic evolution of glioma microenvironment cells based on GIM through sequencing technology at the cell level, expound the evolution of microenvironment cells from the molecular perspective, screen the key mechanisms and targeted molecules of glioma cell crosstalk in the glioma microenvironment, and further confirm the multidimensional regulatory role of targets through a series of in vivo and in vitro experiments. At the same time, we will apply the animal model of glioma for simulating the evolution process. Two-photon micro-fluorescence imaging technology will also be applied for the visual dynamic tracking in the target molecules and microenvironment cells in vivo, revealing the evolution process of glioma microenvironment from a macro-perspective.

恶性胶质瘤的高度异质性和免疫抑制性微环境导致单一分子靶向或免疫治疗起效受限。研究证实胶质瘤相关性小胶质/巨噬细胞（GIM）能和肿瘤细胞相互作用，激活癌通路并调控免疫微环境。我们前期研究发现GIM能与胶质瘤干细胞发生对话，诱导胶质瘤干细胞发生逃逸；同时GIM以通过B7-H4分子营造一个抑制性的免疫微环境。由此，我们推测：针对GIM挖掘能与胶质瘤细胞相互沟通的关键靶点和通路，或可实现同时抑制癌通路和逆转免疫微环境的“靶向免疫治疗”策略。本项目计划以GIM为切入点，在细胞层面进行测序，建立一个胶质瘤微环境细胞动态演进数据库，从分子特征角度阐述微环境细胞的演进，并筛选微环境细胞间的沟通机制和关键靶点，以一系列体内外实验证实靶点的多维度调控作用；同时，利用已构建的胶质瘤动物模型来模拟肿瘤演进过程，应用双光子显微成像技术对靶点和微环境细胞开展活体可视化动态跟踪，从宏观角度揭示胶质瘤微环境的演进过程。

脑胶质瘤是最常见的颅内原发恶性肿瘤，胶质瘤相关性小胶质/巨噬细胞（glioma infiltrating macrophage/microglia，GIM）是免疫微环境中占比最高的免疫细胞，且与肿瘤细胞存在重要的交互行为，是开发肿瘤免疫治疗新策略的重要研究对象。本项目拟通过全转录组、全外显子测序等高通量测序技术获得GIM细胞表达谱数据，筛选重要靶点，并通过可视化技术对靶点和免疫微环境特征进行可视化呈现并尝试临床应用。自立项以来，本项目按照原计划依次完成了GIM-肿瘤细胞互作数据队列的建立、靶点分子免疫调控功能的细胞和动物实验、靶点分子和免疫微环境参与的肿瘤演进特征可视化分析等研究内容，获得以下研究结果： 1）以B7-H4为核心，建立了一种全新的脑胶质瘤免疫三分型，其中与B7-H4相关的免疫亚型极度缺乏免疫细胞的浸润，表现为“超冷”肿瘤；2）通过细胞和动物实验先后证实了两个重要靶点的免疫学功能和分子机制：胶质瘤微环境中的B7-H4通过AKT/eNOS通路抑制CD8+T细胞的免疫功能；STAP1通过Cofilin/F-actin通路抑制GIM的吞噬功能；3) 利用影像组学技术成功建立了一个能准确预测GIM细胞浸润丰度的影像组学模型（RIB模型），同时利用临床试验数据证实了RIB能准确区分DC疫苗免疫治疗获益人群，可成为免疫治疗领域潜在的无创标记物；4）开始建立华山脑肿瘤免疫图谱计划（Brain Cancer Immunology Atlas, BCIA），用于脑肿瘤免疫微环境演进机制和可视化研究，同时推进临床应用。