Mechanisms Promoting Angiogenesis in Glioblastoma

描述(申请人提供)：血管生成是胶质母细胞瘤肿瘤(GBM)的一个显著特征，被认为是一个有前途的治疗靶点。然而，目前可用的抗血管内皮生长因子治疗并不能改变总的存活率，也没有解决分子亚型间血管生成的潜在差异的问题。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，它们至少部分地通过促进内皮细胞(ECs)的迁移来做到这一点。与从正常脑分离的ECs相比，从GBM分离的ECs表现出极大的迁移能力，这种增强的运动性被GSC-CM促进。GSC-CM对内皮细胞的影响似乎部分与促血管生成分子ePhin-B2在ECs上的表达上调有关，这是基于阵列分析和使用重组ePhin-B2-Fc和EphB4-Fc(ePhrin-B2受体)进行的阻断研究。这表明，GSC分泌的因子，包括血管内皮生长因子，通过上调eparin-B2发挥作用。此外，数据表明，GSCs和ECs之间的直接相互作用进一步促进了EC的运动，这种相互作用部分是通过ECs上的整合素v3和GSCs上的L1细胞黏附分子(L1CAM)之间的相互作用来介导的。值得注意的是，由于ewitin-B2促进VEGFR2的激活和信号传递，以及VEGFR2和整合素v3的相互激活，这两种机制可以交叉，从而间接激活整合素v3。因此，我们提出了一个假说模型，在该模型中，GSC分泌的因子上调EC上的促血管生成分子，通过增加EC的运动性来促进GBM中的血管生成，这一机制增强了L1CAM对GSC的直接作用和对ECs的整合素？v？3的作用。这一假说将通过从间充质和前神经亚型的GBM分离的GSC和ECs进行验证，使用重组蛋白、抗体和下调方法：(1)确定GSC分泌的因子(包括血管内皮生长因子)是否促进EC在ECs上的迁移、增殖和管状形态的形成以及促血管生成分子(包括ePhin-B2和EphB4)的表达。(2)确定整合素v与GSCs上的L1CAM的相互作用是否传递促进EC迁移、增殖和/或管状形态形成的信号，通过ECs和GSCs的共同培养；GSC-CM刺激；并将ECs和GSCs注射到脑片后，进行双光子激光扫描显微镜和单个细胞运动跟踪。(3)通过使用PDGF-B驱动的GFP-GBM细胞或在免疫活性整合素3-突变体或对照小鼠脑中增殖的shL1CAM-GFP-GBM细胞来确定ECs和GSCs上整合素和L1CAM的缺失/阻断是否改变血管生成、肿瘤体积和/或存活率，以在体内测试其机制。相关性：这些研究有可能改变对肿瘤相关血管生成的一般理解，并将为改进GBM的抗血管生成治疗提供新的治疗方案。

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.