胶质瘤外泌体miR-21通过正反馈环路增强MSC促进MDSC免疫抑制能力的机制研究

As the most common malignant tumor in central nervous system, glioma responds poorly to traditional treatment. Although novel immunotherapies have been applied to glioma treatment, immuno-suppressive microenvironment of glioma, which contains the Myeloid Derived Suppressor Cells (MDSCs) plays important roles in the resistance to the treatments of glioma. The regulating function of glioma-derived exosome on immune cells impelled us to find new ideas investigating the interactions between glioma cells and immune cells like MDSCs. Recently, we first reported that exosomes derived from hypoxic glioma cells promoted the expansion and immunosuppression capacity of MDSCs through miR-21 dependent pathway. Through further study we found that tumor associated Mesenchymal Stem Cells (MSCs) play an important role in magnifying the signals of miR-21 in exosomes. Hereby, we hypothesized that hypoxic glioma-associated MSCs could promote the expansion and immunosuppression capacity of MDSCs through exosomal miR-21 mediated, multiple positive feedback loops dependent network. We aim to fully elucidate the hypothesis through in vitro and in vivo experiments, combining nano-microfluidic cell culture and microscale exosomes separation-detection technologies, utilizing the causal inference network model to analyze the relevant pathways and molecular mechanisms. Finally we employ the conditional knockout tumor bearing mouse model to design and evaluate the therapeutic regimen based on key points and provide new theoretical basis for modifying the current glioma immunotherapy.

胶质瘤是中枢神经系统最常见的恶性肿瘤，传统疗法效果差。虽然多种免疫治疗策略相继问世，但胶质瘤抑制性免疫微环境中浸润的MDSC等被认为是限制其疗效的重要因素。胶质瘤相关外泌体调控途径为研究胶质瘤细胞与MDSC间细胞通讯和调控提供了新思路。我们前期首次报道了低氧诱导的胶质瘤外泌体能通过miR-21等多个miRNA相关通路促进MDSC增殖和免疫抑制能力。进一步研究发现，胶质瘤相关间充质干细胞可能在上述过程中具有显著放大外泌体miR-21信号的关键介导作用。据此我们提出了由外泌体miR-21介导的、MSC正反馈多环路依赖的低氧胶质瘤促进MDSC增殖及免疫抑制能力的网络调控新假说。本课题拟结合体内外实验及纳米微流控单细胞技术，通过因果推断网络模型深度解析各相关通路效应和分子机制，全面阐明该假说，进而利用条件敲除荷瘤小鼠模型针对关键节点设计并评价干预方案，为改善现有胶质瘤免疫治疗策略提供新的理论依据。

胶质瘤是中枢神经系统最常见的恶性肿瘤，传统疗法效果差。虽然多种免疫治疗策略相继问世，但胶质瘤抑制性免疫微环境中浸润的MDSC等被认为是限制其疗效的重要因素。胶质瘤相关外泌体调控途径为研究胶质瘤细胞与MDSC间细胞通讯和调控提供了新思路。我们前期首次报道了低氧诱导的胶质瘤外泌体能通过miR-21等多个miRNA相关通路促进MDSC增殖和免疫抑制能力。进一步研究发现，胶质瘤相关间充质干细胞可能在上述过程中具有显著放大外泌体miR-21信号的关键介导作用。据此我们提出了由外泌体miR-21介导的、MSC正反馈多环路依赖的低氧胶质瘤促进MDSC增殖及免疫抑制能力的网络调控新假说。通过本研究，我们发现GA-MSC通过外泌体miR-21上调MDSC表面CD73的表达促进胶质瘤进展，而胶质瘤外泌体CD44通过促进转录因子XBP1s的表达上调MSC中的miR-21。同时，MSC中上调的miR-21可通过miR-21/SP1/DNMT1途径降低自身启动子区的DNA甲基化水平，进一步促进miR-21的转录， 形成正反馈环路。胶质瘤外泌体CD44启动的miR-21/SP1/DNMT1正反馈环路可使MSC外泌体miR-21升高至胶质瘤外泌体的十几倍，产生放大胶质瘤外泌体免疫抑制信号的作用，揭示了MSC在胶质瘤微环境中放大外泌体免疫抑制信号的重要作用。