Brain Endothelial TNF-R1 Can Function to Inhibit Angiogenesis

描述(由申请人提供)：通过诱导细胞凋亡来消除新生血管的抗血管生成方法将代表着胶质母细胞瘤治疗的重大进步。肿瘤坏死因子α(TNFa)通过诱导原代培养的人脑微血管内皮细胞(MVEC)表达肿瘤坏死因子受体1(TNF-R1)，从而诱导MVEC凋亡。免疫组织化学分析表明，在大多数患者中，胶质母细胞瘤肿瘤内皮细胞中肿瘤坏死因子-R1和肿瘤坏死因子α的表达明显高于正常脑内皮细胞，而在肿瘤中，肿瘤相关血管生成的水平在肿瘤中是通过注射和增殖小鼠恶性胶质瘤细胞在小鼠脑白质中显著高于野生型小鼠。基于这些和其他数据，我们假设，与恶性胶质瘤相关的脑内皮细胞上肿瘤坏死因子-R1的上调表达是宿主对肿瘤的抗血管生成反应，并且针对肿瘤内皮细胞的TNFa治疗将抑制肿瘤血管生成和肿瘤生长。我们建议通过确定细胞表面信号事件来验证这些假设，这些信号事件可以诱导和调节肿瘤坏死因子-R1介导的胶质母细胞瘤微血管内皮细胞的凋亡。同时，我们将建立以肿瘤微血管内皮细胞为靶点的TNFa融合蛋白与CD13结合肽融合的治疗操作的可行性。我们将使用两种小鼠模型来分析这些效应的特异性和体内反应的幅度：一种是免疫活性的胶质母细胞瘤小鼠模型，另一种是基于人胶质母细胞瘤干细胞的异种移植模型。我们将：(1)利用胶质母细胞瘤患者和正常脑组织的活检，确定在脑瘤MVEC中是否有肿瘤坏死因子-R1的优先表达，并与可能调节其信号调亡能力的分子共定位；(2)确定肿瘤坏死因子-R1是否在脑内作为抗血管生成分子起作用，以应对恶性胶质瘤，并利用肿瘤坏死因子-R1缺失、肿瘤坏死因子1缺失和肿瘤坏死因子-2缺失的小鼠，确定肿瘤坏死因子-R2是否参与或调节这种效应；(3)确定脑MVEC上整合素av？3的激活状态或表达是否调节这些细胞对TNF1促死亡信号的反应；以及(4)检测针对肿瘤内皮细胞表面CD13的TNFa融合蛋白与Cys-Asn-Gly-Arg-Cys肽在体内抑制肿瘤血管生成和肿瘤生长，并促进生存的能力。相关性：结果应该确定一种新的抗血管生成疗法，该疗法可以与其他疗法结合使用，以更有效地消除恶性胶质瘤并防止其复发。这些研究还将提供有关生物标记物的数据，这些生物标记物可用于预测哪些胶质母细胞瘤患者可能从该策略中受益，以及用于非侵入性监测其疗效的生物标记物。

DESCRIPTION (provided by applicant): Anti-angiogenic approaches that eliminate the neovasculature by inducing apoptosis would represent a significant advance in the treatment of glioblastomas. Tumor necrosis factor a (TNFa) can act to induce apoptosis of cultured primary human brain microvessel endothelial cells (MvEC) through a mechanism that requires expression of the TNF-receptor 1 (TNF-R1) on the MvEC. Immunohistochemical analysis of biopsies indicates that, in most patients, the expression of TNF-R1 and TNFa is significantly higher in the glioblastoma tumor endothelial cells as compared to the normal brain endothelial cells and the levels of tumor-associated angiogenesis in tumors developed by injection and propagation of mouse malignant glioma cells in the white matter of the mouse brain is significantly higher in TNF-R1-null mice than in their wild-type counterparts. Based on these and other data, we hypothesize that the upregulated expression of TNF-R1 on brain endothelial cells associated with malignant glioma tumors is a host anti-angiogenic response to the tumor, and that TNFa therapy targeted to tumor endothelial cells will inhibit tumor angiogenesis and tumor growth. We propose to test these hypotheses by identifying the cell surface signaling events that elicit, and regulate, TNF-R1-mediated apoptosis in glioblastoma MvECs. In parallel, we will establish the feasibility of therapeutic manipulation of TNF-R1 with a TNFa fusion protein that is targeted to tumor MvECs by fusion with a peptide that binds CD13. We will use two mouse models to analyze the specificity of the effects and the magnitude of the responses in vivo: an immune competent mouse model of glioblastoma and a xenograft model based on the use of human glioblastoma stem cells. We will: (1) Establish whether TNF-R1 is preferentially expressed in the brain tumor MvEC and is colocalized with molecules that may regulate its ability to signal apoptosis, using biopsies from patients with glioblastoma and normal brain; (2) Determine whether TNF-R1 functions as an anti-angiogenic molecule in the brain in response to a malignant glioma tumor and establish whether TNF- R2 contributes to, or modulates, this effect using TNF-R1-null, TNF1-null, and TNF-R2-null mice; (3) Determine whether the activation state or expression of integrin av¿3 on the brain MvEC modulates the response of these cells to the pro-death signaling of TNF1; and (4) Test the ability of a TNFa fusion protein targeted to CD13 on tumor endothelial cells with the Cys-Asn-Gly-Arg-Cys peptide to inhibit tumor angiogenesis and tumor growth, and to promote survival, in vivo. RELEVANCE: The results should identify a novel anti-angiogenic therapy that can be used in conjunction with other therapies to more effectively eliminate malignant glioma tumors and prevent their recurrence. The studies also will provide data concerning biomarkers that may be used to predict which glioblastoma patients may benefit from this strategy and biomarkers for non-invasive monitoring of its efficacy.