Mechanisms Promoting Angiogenesis in Glioblastoma

促进胶质母细胞瘤血管生成的机制

• 批准号: 9233971
• 负责人: Candece L Gladson
• 金额: $ 34.39万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9233971
• 关键词: Address; Alpha Cell; Angiogenic Factor; Antibodies; Antibody Therapy; Apoptosis; Binding; Biological Assay; Blocking Antibodies; Brain; Cell Death; Cell Proliferation; Cells; Characteristics; Coculture Techniques; Confocal Microscopy; Data; Down-Regulation; Endothelial Cells; Eph Family Receptors; EphB4 Receptor; Ephrin-B2; Exhibits; Glioblastoma; Glioma; Height; Immune; Immunocompetent; In Situ; Individual; Injection of therapeutic agent; Integrins; KDR gene; Laser Scanning Microscopy; Link; Malignant Neoplasms; Mediating; Mesenchymal Cell Neoplasm; Messenger RNA; Modeling; Mus; Mutation; Neural Cell Adhesion Molecule L1; Patients; Peptides; Platelet-Derived Growth Factor; Play; Process; Proliferation Marker; Quantitative Reverse Transcriptase PCR; Recombinant Proteins; Recombinants; Regimen; Signal Transduction; Slice; Stem cells; Testing; Tumor Angiogenesis; Tumor Stem Cells; Tumor Volume; Up-Regulation; Vascular Endothelial Growth Factors; angiogenesis; antiangiogenesis therapy; base; blood vessel development; brain endothelial cell; cell motility; improved; in vivo; knock-down; migration; migration stimulating factor; molecular subtypes; mutant; neoplastic cell; neutralizing antibody; novel; personalized therapeutic; public health relevance; receptor; relating to nervous system; therapeutic target; transcriptome; tumor; two-photon

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.

描述（由适用提供）：血管生成是胶质母细胞瘤肿瘤（GBM）的突出特征，被认为是有前途的治疗靶标。但是，目前可用的抗VEGF疗法并未改变总体生存率，并且尚未解决GBM分子亚型中血管生成的潜在差异问题。我们的初步数据支持了胶质瘤干细胞（GSC）在与GBM相关的血管生成中起关键作用的概念，并且至少部分通过增强内皮细胞（ECS）的迁移来做到这一点。与从正常大脑分离的EC相比，从GBM中分离出的EC非常增强，并且通过GSC条件培养基（GSC-CM）促进了这种高度运动。 GSC-CM对EC的影响似乎部分与基于阵列分析和使用重组Ephrin-B2-FC和EphB4-FC（Ephrin-B2受体）的阵列分析和阻断研究的前血管生成分子Ephrin-B2对ECS的上调有关。这表明，包括VEGF在内的GSC分泌的因素通过上调ephrin-b2来起作用。此外，数据表明GSC与EC之间的直接相互作用进一步促进了EC运动，并且这种相互作用部分是由于ECS上的整联蛋白与GSC上的L1细胞粘附分子（L1CAM）之间的相互作用介导的。值得注意的是，由于ephrin-b2促进了vegfr2的激活和信号传导，而随着vegfr2和整合素的相互作用相互激活，这两种机制可能会相交，从而导致ephrin-b2间接激活整联蛋白。这是我们提出的一个假设模型，在该模型中，GBM中的血管生成是由GSC分泌因子驱动的增加的EC运动促进的，该因子驱动了eC的促血管生成分子对EC的上调，并且该机制增强了L1CAM对GSCS对GSCS和ECS的L1CAM之间直接相互作用的影响。 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）在EC上。 （2）确定整联蛋白在EC上与GSC上L1CAM的相互作用是否通过通过ECS和GSC的共培养确定的促进EC迁移，增殖和/或微型形态发生的信号传递。用GSC-CM刺激；将EC和GSC注入大脑切片中，然后将2光子激光扫描显微镜和单个细胞运动跟踪。 (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 脑。相关性：这些研究有可能在对肿瘤相关的血管生成的理解方面提供范式转移，并建议改善GBM抗血管生成治疗的新方案。