Brain Endothelial TNF-R1 Can Function to Inhibit Angiogenesis

描述（由申请方提供）：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α（TNF α）可以通过需要在MvEC上表达TNF受体1（TNF-R1）的机制来诱导培养的原代人脑微血管内皮细胞（MvEC）的凋亡。活检的免疫组织化学分析表明，在大多数患者中，与正常脑内皮细胞相比，在胶质母细胞瘤肿瘤内皮细胞中TNF-R1和TNF α的表达显著更高，并且在通过注射小鼠恶性神经胶质瘤细胞并使其在小鼠脑的白色物质中增殖而形成的肿瘤中，肿瘤相关血管生成的水平在TNF-R1中显著更高。null小鼠比它们的野生型对应物。基于这些和其他数据，我们假设与恶性神经胶质瘤肿瘤相关的脑内皮细胞上TNF-R1的上调表达是宿主对肿瘤的抗血管生成反应，并且靶向肿瘤内皮细胞的TNFa治疗将抑制肿瘤血管生成和肿瘤生长。我们建议通过识别引发和调节胶质母细胞瘤MvEC中TNF-R1介导的凋亡的细胞表面信号事件来测试这些假设。与此同时，我们将建立TNF-R1与TNFa融合蛋白的治疗操作的可行性，该TNFa融合蛋白通过与结合CD 13的肽融合而靶向肿瘤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幅度：胶质母细胞瘤的免疫活性小鼠模型和基于使用人胶质母细胞瘤干细胞的异种移植模型。我们将：（1）使用来自胶质母细胞瘤患者和正常脑的活组织检查，确定TNF-R1是否优先在脑肿瘤MvEC中表达，并与可能调节其信号细胞凋亡能力的分子共定位;（2）确定TNF-R1是否在脑中作为抗血管生成分子响应恶性神经胶质瘤肿瘤发挥作用，并确定TNF-R2是否有助于或调节，使用TNF-R1-null、TNF 1-null和TNF-R2-null小鼠的这种效应;（3）确定脑MvEC上的整合素α v β 3的活化状态或表达是否调节这些细胞对TNF 1的促死亡信号的应答;和（4）测试靶向肿瘤内皮细胞上的CD 13的TNF α融合蛋白与Cys-Asn-Gly-Arg-Cys肽抑制肿瘤血管生成和肿瘤生长的能力，并促进体内存活。相关性：结果应该确定一种新的抗血管生成疗法，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受益于这种策略，以及用于非侵入性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.