Host-mediated targets in glioma invasion

描述（由申请人提供）：恶性胶质瘤生长的特点是肿瘤广泛浸润并伴有血脑屏障（BBB）的破坏。为了确定血脑屏障破坏减少对胶质瘤浸润的影响，我们开发了一种原位异种移植小鼠脑瘤模型，将胶质瘤植入免疫缺陷Src敲除小鼠（Src KO）。Src KO小鼠先前已被证明可以介导胶质瘤诱导的血脑屏障破坏的减少，这与胶质瘤浸润的减少有关，但一般不直接影响胶质瘤的生长。在这个模型中，重点是宿主细胞室的影响（即血管内皮中的Src缺陷），而不是更常见的肿瘤细胞本身（即肿瘤细胞室）的分析。拟议的研究响应PAS（“理解脑肿瘤扩散机制”），概述了一种新的蛋白质组学策略，以识别和定量Src介导的表达和磷酸化变化，以响应胶质瘤介导的Src KO与野生型（WT）小鼠大脑中的血脑屏障分解。与RNA分析相比，蛋白质组学能够检测蛋白质表达的变化，而不会引入不同mrna的可变翻译效率/稳定性的偏见。在Aim 1中，内皮细胞将从荷瘤Src KO和WT小鼠脑中分离出来，以鉴定Src调节蛋白。对于在Aim 1中鉴定的每个src介导的候选靶标分子，我们将在Aim 2中验证候选靶标在血脑屏障重建试验中的功能。这些研究的价值在于鉴定与胶质瘤侵袭相关的血脑屏障（BBB）蛋白表达变化，使用具有明确表型的敲除模型：1)减少vegf诱导的血脑屏障破坏；2)减少胶质瘤诱导的血脑屏障破坏；3)减少胶质瘤侵袭；4)减少胶质瘤诱导的血管周围纤维蛋白（原）积累。这种异种移植物/蛋白质组学方法是一种新颖的策略，对肿瘤-宿主相互作用具有更广泛的意义，因为它可以在肿瘤诱导的肿瘤微环境重塑中识别宿主来源（即小鼠）和肿瘤来源（即人类）的蛋白质。公共卫生相关性：这些研究通过分析介导血脑屏障破坏的血管中蛋白质表达的变化来解决PAS-06-201（理解脑肿瘤扩散机制）。这些蛋白质的鉴定将有助于更好地理解调节脑肿瘤扩散的机制，并有助于未来设计针对宿主而非肿瘤细胞本身的创新疗法。

DESCRIPTION (provided by applicant): Malignant glioma growth is characterized by extensive tumor infiltration associated with breakdown of the blood brain barrier (BBB). To determine the effect of reduced BBB breakdown on glioma infiltration we have developed an orthotopic xenograft mouse brain tumor model in which gliomas are implanted into immunodeficient Src knockout mice (Src KO). The Src KO mouse has been previously shown to mediate a reduction in glioma-induced BBB breakdown that was associated with reduced glioma infiltration, but independent of direct effects on glioma growth in general. In this model the focus is on effects of the host compartment (i.e. Src defects in the vascular endothelium) rather than on the more common analysis of tumor cells themselves (i.e. tumor compartment). The proposed studies are responsive to the PAS ("Understanding mechanisms of brain tumor dispersal") by outlining a novel proteomics strategy to identify and quantitate Src-mediated changes in expression and phosphorylation in response to glioma-mediated BBB breakdown in Src KO vs. wild type (WT) mouse brains. In contrast to RNA analyses, proteomics enables the detection of changes in protein expression without introducing a bias from variable translational efficiency/stability of different mRNAs. In Aim 1 endothelial cells will be isolated from tumor-bearing Src KO and WT mouse brains to identify Src-regulated proteins. For each candidate Src-mediated target molecule identified in Aim 1, we will validate the function of a candidate hit in a BBB reconstitution assay in Aim 2. The value of these studies is the identification of protein expression changes in the blood brain barrier (BBB) associated with glioma invasion using a knockout model with a well-defined phenotype exhibiting: 1) reduced VEGF-induced breakdown of the BBB; 2) reduced glioma-induced breakdown of the BBB; 3) reduced glioma invasion; and 4) reduced glioma-induced perivascular fibrin(-ogen) accumulation. This xenograft/proteomics approach is a novel strategy with much wider implications for tumor-host interactions, since it enables the identification of host-derived (i.e. mouse) vs. tumor-derived (i.e. human) proteins in the tumor-induced remodeling of the tumor microenvironment. PUBLIC HEALTH RELEVANCE: These studies address the PAS-06-201 (Understanding mechanisms of brain tumor dispersal) by analyzing changes in protein expression in blood vessels that mediate the breakdown of the blood brain barrier. The identification of these proteins will lead to a better understanding of the mechanisms regulating brain tumor dispersal and the future design of innovative therapies that target the host rather than tumor cells themselves.