胶质瘤干细胞在血管拟态形成中的作用及其机制研究

Not only the lack of comprehensive understanding of the underlying mechanism for the formation of newly discovered vasculogenic mimicry (VM), a special type of microvessels lined by cancer cells other than vascular endothelia, but also none of effective inhibitory regimens to specifically target VM available for clinicians are major reasons that current conventional anti-angiogenesis therapies are less effective and even more often failed clinically. Our previous studies demonstrate that cancer stem cells (CSCs) not only contribute to angiogenesis but also give rise to endothelial cells and participate in the VM formation of which VEGFR-2 and Twist1 may play an important role. This study will focus on investigating the roles and mechanisms of CSCs in the formation of VM. Using CSCs derived from glioma to establish several in vivo and in vitro new models for VM study, we will employ several technologies available in our group including live cell tracking, two-photon and laser confocal microscopy, laser capture microdissection, and all "-omic" analyses, etc., not only to investigate VM characteristics, development and cellular origin, but also to reveal its phenotype transition (surface markers) and destination, and identify the signaling pathways that control the stemness maintenance and extracellular matrix production in CSCs during the process of VM formation. Finally, we will try to find or screen for inhibitors, agonists and/or antibodies specific for VM-related molecules and/or signaling pathways and examine their efficacies for intervention. The results of this proposed study is expected not only to provide a better understanding for cellular and molecular mechanisms of VM formation and demonstrate its important clinical significance for prognostic evaluation and more accurate pathological diagnosis of cancer, but also to yield novel strategies and regimens for CSCs-based personalized VM-targeted anti-tumor therapy to treat cancer patients.

近年发现的肿瘤非内皮依赖性"血管"(血管拟态，VM)形成机制不清，缺乏阻抑措施，导致抗肿瘤血管生成疗效欠佳。我们前期研究发现，肿瘤干细胞(CSCs)不仅诱导血管生成、转分化为内皮，还是VM形成的关键细胞，其中VEGFR-2和Twist1可能起重要作用。本项目围绕"CSCs在VM形成中的作用及机制"这一关键科学问题，以人脑胶质瘤CSCs及其移植瘤为主要材料，创建一系列体外和在体VM研究模型，采用活细胞示踪、双光子与共聚焦显微术、显微切割与组学检测等技术，进一步研究VM特征、形成规律和细胞来源，明确CSCs表型转化(标志物)和归宿，认识VM形成中CSCs"干性"维持、基质分泌相关的主要信号通路，并针对VM的分子靶点，筛选拮抗剂、抑制剂或制备单抗，观测干预效应。研究结果对阐明肿瘤VM形成的细胞与分子机制及其诊断价值具有重要意义，并将为基于CSCs的抗VM个体化肿瘤治疗提供新策略、新方法。

近年发现的肿瘤内非内皮依赖性“血管”（血管拟态）形成机制不清，缺乏阻抑措施，导致抗肿瘤血管生成治疗疗效欠佳。基于前期研究结果，拟以胶质瘤为研究模型，围绕“GSCs在VM形成中的作用及其分子机制”这一关键问题开展深入研究，发现，VM密度高的胶质母细胞瘤病人的总生存期和无进展生存期均较VM数量低组短，预后差；VEGFR-2活化可促进GSCs迁移和体外管型样结构的形成（VM）；阻断或下调VEGFR-2后，GSCs失去了在体外形成管型样结构的能力；将VEGFR-2-shRNA的GSCs移植入裸鼠体内，发现GSCs失去在体内形成VM的能力。采用贝伐单抗中和VEGF后，GSCs仍能形成VM样结构，同时自噬活性增强；在裸鼠原位移植瘤模型中给予抗血管药物贝伐单抗治疗后血管密度显著降低，血管拟态数量明显增多，自噬蛋白ATG5表达上调；自噬通过非VEGF依赖的ROS途径激活VEGFR-2-Y1175位点，介导细胞形成VM；自噬抑制剂氯喹（Chloroquine）联合贝伐单抗治疗可显著抑制肿瘤生长，延长荷瘤鼠的生存期。进一步的研究发现GSCs可分化为血管周细胞，通过分析患者生存期发现，血管周细胞覆盖率高的患者预后较差，患者生存时间与血管周细胞覆盖率呈负相关。进一步将胶质母细胞瘤患者分为接受化疗组和未接受化疗组，发现在接受化疗的患者中，患者生存期与血管周细胞覆盖率呈显著负相关，血管周细胞覆盖率越高，患者预后越差。而在未接受化疗组，患者生存期与血管周细胞覆盖率无关。针对胶质瘤干细胞源性周细胞的治疗研究发现，靶向清除胶质瘤源性血管周细胞能够增加血管的弥散性，dextran (10 kDa)可以透过血瘤屏障到达肿瘤部位，而对照组dextran无法通过血瘤屏障，提示靶向清除胶质瘤源性血管周细胞可通过增加血管弥散性而提高化疗效果。Etoposide曾被用于胶质瘤治疗，后因其不容易透过血瘤屏障而被停用。我们发现特异性去除GSC源性周细胞后，Etoposide对胶质瘤原位移植瘤显示出良好治疗效果，能够显著抑制移植瘤生长，延长裸鼠生存期。本项目首次揭示了胶质瘤内血管拟态样结构的细胞来源，阐明了其分子机制，并明确了靶向胶质瘤干细胞源性血管拟态/血管周细胞的治疗学意义。