PDGF诱导胶质母细胞瘤抗血管靶向治疗耐药及其分子机制研究

Glioblastoma (GBM, grade IV glioma) is the most malignant primary brain tumor. High vasculature of GBM indicates the tumor vessel is an important target for GBM therapy. However, most malignant solid tumors including GBM exhibit robust resistance to current anti-angiogenic therapies that primarily target VEGF pathways. Recently, we systemically investigated the mechanisms of tumor development induced by abnormality of vessels within the tumor （Nature Cell Biology 2014; J Clin Invest 2016), suggesting that PDGF could mediate tumor resistance against anti-VEGF therapy. In current proposal, we preliminarily show that RNA-seq analysis identifies reduced VEGFR2 expression in GBM ECs and shows increased mesenchymal gene expression such as FSP-1. Analyzing human GBM-derived endothelial cells (ECs) and EC lineage-derived cells in a genetic murine GBM model, we confirm that VEGFR2 expression is reduced in GBM ECs. Therefore, we demonstrate endothelial mesenchymal transformation (Endo-MT) induces GBM resistance to anti-angiogenic therapy by downregulating VEGFR2 expression. We will try to further identify molecular mechanism that induces mesenchymal transformation and reduces VEGFR2 expression. Finally, dual inhibition of VEGFR and PDGFR will be performed to verify if elimination of appropriate signaling pathways within the tumor-associated ECs improves animal survival in GBM-bearing mice. EC-specific knockout of PDGFR will be carried out to verify the sensitization of tumors to VEGF-neutralizing antibody treatment. We expect that these studies will be able to reveal an endothelial plasticity-mediated mechanism controlling anti-angiogenic therapy resistance, and the combination of vascular de-transformation and angiogenic factor blockade may offer promising opportunities for anti-vascular therapy in GBM and possibly other solid tumors.

胶质母细胞瘤（GBM，即IV级胶质瘤）是恶性度最高的原发脑肿瘤。GBM高度血管化提示其肿瘤血管可作为重要的治疗靶点。然而最新临床试验表明GBM普遍对抗血管靶向治疗。最近我们系统研究了血管异常化驱动肿瘤发展的机制 （Nature Cell Biology 2014; J Clin Invest 2016），提示PDGF通路可能诱导了肿瘤对抗VEGF治疗的耐药性。在本项目中，我们利用GBM细胞模型探讨PDGF是否通过下调内皮细胞VEGFR2和上调FSP-1来介导血管异常化和抗血管治疗耐药性及其作用分子机制；使用GBM病人来源血管内皮细胞和组织样本验证PDGF调控的基因表达和遗传学机制。最后我们将使用自己构建的条件性PDGFR敲除小鼠和基因工程诱导的GBM动物模型来验证特异性的PDGF和/或VEGF通路靶向抑制的治疗作用。我们期望揭示GBM抗血管靶向治疗耐药新机制，指导新药开发和新型联合用药。

在经费下达后，基本按时完成了项目的主要内容，整个研究主要包括三部分：1）体外细胞模型研究：采用体外GBM条件培养基培养人脑微血管EC模型并收集RNA和总蛋白行高通量全基因组RNA测序（RNA-seq）来筛选GBM条件培养基上调或下调的关键基因并鉴定出关键的PDGF和FSP-1上调、VEGFR2下调。使用多种PDGF因子、PDGF 中和抗体、PDGFRɑ/ß-siRNA敲低、PDGFRɑ/ß 特异性抑制剂和VEGF中和性抗体B20、VEGFR特异抑制剂Ki8751等来进一步评价PDGF是否调控及如何调控VEGFR2和FSP-1的表达水平；PDGF是否通过调控VEGFR2和FSP-1来诱导内皮细胞抗血管靶向治疗的耐受性。2）体内动物模型研究：运用基因工程技术构建鼠转基因GBM模型，分离出GBM中内皮细胞EC，通过流式细胞定量技术检测EC VEGFR2的表达；构建条件性 PDGFR 敲除小鼠，结合VEGFR和/或PDGFR的多种特异性抑制剂来单独或联合治疗鼠 GBM，评价PDGF和/或 VEGF通路特异性靶向抑制GBM的治疗作用。3）人体标本研究：最后运用临床GBM病例，分离人脑GBM肿瘤中、肿瘤周边和周围正常CD31+EC原代细胞，收集蛋白质行免疫印迹分析VEGFR2的表达差异；采用VEGF特异抗体B20和VEGFR特异抑制剂Ki8751来分别处理EC细胞并比较和评价其存活力影响等生物学功能。除人体标本研究因样本量不足暂时未能完成外，其他研究内容均已顺利实施，接下来将继续收集标本进一步研究。.根据研究计划及实际研究调整，研究工作集中在：1）PDGF介导的间充质转化导致胶质母细胞瘤内皮细胞VEGF耐药的机制研究；2）氧化维生素C（DHA）通过干扰能量平衡克服EGFR靶向治疗癌症的耐药性研究；3）微波消融重建肿瘤微环境增强AXL特异性CAR T细胞对非小细胞肺癌的免疫治疗；4）分泌IL-7和CCL19的CAR-T细胞治疗GPC3/Mesothelin阳性的肿瘤；5）烟酰胺核糖通过调节Smads信号通路的乙酰化来保护CCl4诱导的肝纤维化。