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治疗并不能改变总生存率，并且GBM分子亚型之间血管生成的潜在差异问题尚未得到解决。我们的初步数据支持胶质瘤干细胞（GSCs）在gbm相关血管生成中发挥关键作用的概念，至少部分是通过增强内皮细胞（ECs）的迁移来实现的。与从正常脑中分离的脑外细胞相比，从GBM中分离的脑外细胞表现出大大增强的迁移能力，这种高度运动性是由gsc条件培养基（GSC-CM）促进的。基于阵列分析和重组ephrin-B2- fc和EphB4-Fc （ephrin-B2受体）的阻断研究，GSC-CM对内皮细胞的影响似乎部分与促血管生成分子ephrin-B2对内皮细胞的上调有关。这表明GSCs分泌的因子，包括VEGF，通过上调ephrin-B2起作用。此外，数据表明，GSCs和EC之间的直接相互作用进一步促进了EC的运动，并且这种相互作用部分是由EC上的整合素¿v¿3和GSCs上的L1细胞粘附分子（L1细胞粘附分子）之间的相互作用介导的。值得注意的是，由于ephrin-B2促进VEGFR2的激活和信号转导，以及VEGFR2和整合素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3。因此，我们提出了一个假设模型，其中gsc分泌的因子上调EC上的促血管生成分子，从而驱动EC运动增加，从而促进GBM中的血管生成，并且该机制增强了L1CAM对gsc和整合素对EC的直接相互作用的作用。我们将利用重组蛋白、抗体和下调方法，利用从间充质和前神经亚型GBM中分离的GSCs和EC来验证这一假设：(1)确定gsc分泌的因子（包括VEGF）是否促进EC的迁移、增殖和小管形态形成，以及促血管生成分子（包括ephrin-B2和EphB4）在EC上的表达。(2)通过ECs与GSCs共培养，确定ECs上的整合素3与GSCs上的L1CAM的相互作用是否传递了促进EC迁移、增殖和/或微管形态形成的信号；GSC-CM刺激；并将ECs和GSCs注入脑切片，然后进行2光子激光扫描显微镜和单个细胞运动跟踪。(3)该机制将在体内进行测试，通过确定pdgf - b驱动的GFP-GBM细胞或shL1CAM- GFP-GBM细胞在免疫活性整合素- 3突变体或对照小鼠大脑中增殖，是否缺乏/阻断ECs上的整合素- v - 3或ephrin-B2和GSCs上的L1CAM会改变血管生成、肿瘤体积和/或存活。相关性：这些研究有可能为理解肿瘤相关血管生成提供范式转变，并将为改善GBM抗血管生成治疗提供新的方案。

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.