Mechanisms Promoting Angiogenesis in Glioblastoma

描述（由申请人提供）：血管生成是胶质母细胞瘤（GBM）的一个突出特征，被认为是一个有前途的治疗靶点。然而，目前可用的抗VEGF治疗不改变总生存率，并且GBM分子亚型之间血管生成的潜在差异的问题尚未得到解决。我们的初步数据支持这样的概念，即胶质瘤干细胞（GSC）在GBM相关的血管生成中起着关键作用，并且至少部分地通过增强内皮细胞（EC）的迁移来实现。与从正常脑分离的EC相比，从GBM分离的EC表现出极大增强的迁移，并且这种增强的运动性由GSC条件培养基（GSC-CM）促进。基于阵列分析和使用重组肝配蛋白-B2-Fc和EphB 4-Fc（肝配蛋白-B2受体）的阻断研究，GSC-CM对EC的作用似乎部分与EC上促血管生成分子肝配蛋白-B2的上调相关。这表明GSC分泌的因子，包括VEGF，通过上调肝配蛋白-B2起作用。此外，这些数据表明，GSC和EC之间的直接相互作用进一步促进EC运动性，并且这种相互作用部分是由EC上的整合素β 3和GSC上的L1细胞粘附分子（L1 CAM）之间的相互作用介导的。值得注意的是，由于ephrin-B2促进VEGFR 2的激活和信号传导，并且VEGFR 2和整合素β 3相互激活，这两种机制可能交叉，导致ephrin-B2间接激活整合素β 3。因此，我们提出了一个假设的模型，其中GBM中的血管生成是通过增加EC运动性来促进的，EC运动性是由GSC分泌的因子驱动的，这些因子上调EC上的促血管生成分子，并且这种机制增强了GSC上的L1 CAM和EC上的整合素β 3之间的直接相互作用的效果。将使用从间充质和前神经亚型的GBM分离的GSC和EC，使用重组蛋白、抗体和下调方法来测试该假设，以：（1）确定GSC分泌的因子（包括VEGF）是否促进EC迁移、增殖和小管形态发生以及促血管生成分子（包括肝配蛋白-B2和EphB 4）在EC上的表达。(2)确定EC上的整合素β 3与GSC上的L1 CAM的相互作用是否传递促进EC迁移、增殖和/或微管形态发生的信号，如通过EC和GSC的共培养所确定的;用GSC-CM刺激;以及将EC和GSC注射到脑切片中，然后进行双光子激光扫描显微镜检查和单个细胞运动跟踪。(3)该机制将通过使用PDGF-B驱动的GFP-GBM细胞或shL 1CAM-GFP-GBM细胞在免疫活性整合素β 3突变体或对照小鼠脑中繁殖来确定EC上的整合素β 3或肝配蛋白β 2和GSC上的L1 CAM的缺失/阻断是否改变血管生成、肿瘤体积和/或存活来进行体内测试。相关性：这些研究有可能提供一个范式的转变，在一般的理解肿瘤相关的血管生成，并建议新的方案，改善抗血管生成治疗GBM。

DESCRIPTION (provided by applicant): Angiogenesis is a prominent characteristic of glioblastoma tumors (GBM) and is considered a promising therapeutic target. Currently available anti-VEGF therapy does not change overall survival, however, and the issue of potential differences in angiogenesis among the molecular subtypes of GBM has not been addressed. Our preliminary data support the concept that glioma stem cells (GSCs) play a key role in GBM-associated angiogenesis and they do so, at least in part, by enhancing the migration of endothelial cells (ECs). ECs isolated from GBM exhibit greatly enhanced migration as compared to ECs isolated from normal brain and this heighted motility is promoted by GSC-conditioned media (GSC-CM). The effects of GSC-CM on the ECs appear to be associated, in part, with upregulation of the pro-angiogenic molecule ephrin-B2 on the ECs based on array analysis and blocking studies using recombinant ephrin-B2-Fc and EphB4-Fc (the ephrin-B2 receptor). This suggests that factors secreted by GSCs, including VEGF, act by upregulating ephrin-B2. In addition, the data indicate that a direct interaction between GSCs and ECs further promotes EC motility and that this interaction is mediated, in part, by an interaction between integrin �v�3 on ECs and L1 cell adhesion molecule (L1CAM) on GSCs. Notably, as ephrin-B2 promotes the activation and signaling of VEGFR2 and as VEGFR2 and integrin �v�3 reciprocally activate each other, these two mechanisms could intersect resulting in ephrin-B2 indirectly activating integrin �v�3. Thus, we propose a hypothetical model in which angiogenesis in GBM is promoted by increased EC motility that is driven by GSC-secreted factors that upregulate pro- angiogenic molecules on EC and that this mechanism enhances the effects of a direct interaction between L1CAM on the GSCs and integrin �v�3 on the ECs. This hypothesis will be tested using GSCs and ECs isolated from GBM of the mesenchymal and pro-neural subtypes using recombinant proteins, antibodies and downregulation approaches to: (1) Determine whether GSC-secreted factors (including VEGF) promote EC migration, proliferation and tubulomorphogenesis and expression of pro-angiogenic molecules (including ephrin-B2 and EphB4) on ECs. (2) Determine whether the interaction of integrin �v�3 on ECs with L1CAM on GSCs transmits a signal promoting EC migration, proliferation and/or tubulomorphogenesis as determined by co-culture of ECs and GSCs; stimulation with GSC-CM; and injection of ECs and GSCs into brain slices followed by 2-Photon Laser Scanning microscopy and individual cell motility tracking. (3) The mechanism will be tested in vivo by determining if the absence/blocking of integrin �v�3 or ephrin-B2 on ECs and L1CAM on GSCs alters angiogenesis, tumor volume and/or survival using PDGF-B-driven GFP-GBM cells or shL1CAM- GFP-GBM cells propagated in immunocompetent integrin �3-mutant or control mouse brain. RELEVANCE: These studies have the potential to provide a paradigm shift in the understanding of tumor-associated angiogenesis in general and will suggest novel regimens for improved anti-angiogenesis therapy in GBM.