Brain Endothelial TNF-R1 Can Function to Inhibit Angiogenesis

描述（由申请人提供）：通过诱导细胞凋亡消除新生血管的抗血管生成方法将代表胶质母细胞瘤治疗的重大进展。肿瘤坏死因子a （Tumor necrosis factor a, TNFa）可诱导培养的原代人脑微血管内皮细胞（MvEC）凋亡，其机制需要在MvEC上表达tnf -受体1 （TNF-R1）。活检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TNF-R1和TNFa的表达明显高于正常脑内皮细胞，而在小鼠脑白质中注射和增殖小鼠恶性胶质瘤细胞形成的肿瘤中，TNF-R1缺失小鼠的肿瘤相关血管生成水平明显高于野生型小鼠。基于这些及其他数据，我们假设与恶性胶质瘤相关的脑内皮细胞上TNF-R1表达上调是宿主对肿瘤的抗血管生成反应，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。我们建议通过鉴定胶质母细胞瘤MvECs中引发和调节tnf - r1介导的细胞凋亡的细胞表面信号事件来验证这些假设。同时，我们将建立治疗性操作TNF-R1的可行性，该TNFa融合蛋白通过与结合CD13的肽融合靶向肿瘤mvec。我们将使用两种小鼠模型来分析体内效应的特异性和反应的强度：胶质母细胞瘤免疫小鼠模型和基于使用人类胶质母细胞瘤干细胞的异种移植模型。我们将：(1)利用胶质母细胞瘤和正常脑患者的活检，确定TNF-R1是否在脑肿瘤MvEC中优先表达，并与可能调节其凋亡信号能力的分子共定位；(2)利用TNF- r1缺失、TNF- f1缺失和TNF-R2缺失的小鼠，确定TNF- r1是否在脑恶性胶质瘤肿瘤反应中作为抗血管生成分子发挥作用，并确定TNF-R2是否有助于或调节这种作用；(3)确定整合素av¿3在脑MvEC上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；(4)用Cys-Asn-Gly-Arg-Cys肽检测靶向CD13的tnf - fa融合蛋白在肿瘤内皮细胞上抑制肿瘤血管生成和肿瘤生长、促进肿瘤存活的能力。相关性：该结果应该确定一种新的抗血管生成疗法，可以与其他疗法联合使用，更有效地消除恶性胶质瘤肿瘤并防止其复发。这些研究还将提供有关生物标志物的数据，这些生物标志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，并可用于无创监测其疗效的生物标志物。

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.