Plexin-B2 Function in Glioma Invasion

恶性脑肿瘤胶质母细胞瘤(GBM)具有高度浸润性。迁移的GBM细胞暴露在 然而，肿瘤侵袭过程中的生物力学作用，对其机械敏感途径知之甚少。 使基底膜细胞获得侵袭性。在这里，我们假设丛状蛋白引导受体，集中在这个 丛状蛋白-B2及其类似物丛状蛋白-D1的研究(两者在GBM中高表达并与差相关 患者存活率)，可能是增强机械张力诱导的胶质瘤细胞的关键机械调节因子 迁移。我们的假设建立在一系列令人兴奋的最新发现之上：在原位移植模型中， 使用患者来源的GBM干细胞(GSCs)，我们发现Plexin-B2基因敲除(KO)不仅导致 减少了肿瘤的扩散，但也显著地改变了从轴突束到血管周围的迁移偏好 路线。此外，当GSCs在条带分析中同时在软和硬的底物上扩散时，Plexin-B2 KO GSCs 聚集在僵硬的条纹上，这种迁徙行为被称为趋多性。丛状蛋白-B2的赋权能力 基底膜细胞克服趋化倾向具有临床意义：随着基底膜的进展，肿瘤的大部分 逐渐变硬，部分是由于压力的增加；因此，肿瘤细胞必须找到一种方法来脱离肿瘤 大量侵入较软的脑实质，我们的初步数据表明丛状蛋白上调可能满足 这个角色。为了进一步剖析机械张力诱导的基底膜侵袭，我们将研究丛蛋白的机制细节。 B2和-D1作为控制GBM入侵的机制调节器，目的是确定新的目标来遏制 基底膜渗入。在目标1中，我们将扩大体内移植研究，以测试丛状蛋白介导的GBM侵袭 模式和迁移路径适用于不同的GBM亚型。然后，我们将调查差异化如何 状态和代谢生态位(低氧)可能改变依赖于丛状蛋白的迁移路线的选择。我们 将在人类GBM组织中验证这些发现。在目标2中，我们将深入研究Plexin是如何运作以促进 侵犯性。我们将应用一系列的机械敏感性分析来研究网络蛋白介导的生物力学。 基底膜细胞的迁移特性，包括细胞间黏附、细胞分散能力、肌动球蛋白 动力学，以及3D血管模型中的渗透行为。我们将测试迁移的GBM细胞如何反应 不同的底物硬度、基质底物和解离状态，以及丛状蛋白如何改变趋化作用 行为。我们将在GBM细胞中定义机械反应路径，并将它们用作读数来直接测试 丛状蛋白的信号素和机械依赖性功能。最后，在目标3中，我们将询问下游 丛蛋白-B2机械信号转导的效应器。这包括丛状蛋白与机械敏感性的相互作用。 HIPPO/YAP途径，以及通过细胞内蛋白RAP2或AMOT可能的转导机制。总而言之，通过 通过对基底膜侵袭机制的研究，我们探索了基底膜恶性的新范式。 最终目标是寻找针对这种致命癌症的新治疗机会。

The malignant brain tumor glioblastoma (GBM) is highly infiltrative. Migrating GBM cells are exposed to biomechanical forces during tumor invasion, however, little is understood of the mechanosensitive pathways that enable GBM cells to gain invasiveness. Here, we postulate that Plexin guidance receptors, focusing in this proposal on Plexin-B2 and its paralog Plexin-D1 (both highly upregulated in GBM and correlated with poor patient survival), may function as key mechanoregulators to enhance mechanotension-induced glioma cell migration. Our hypothesis is built upon a series of exciting recent discoveries: in orthotopic transplant models, using patient-derived GBM stem cells (GSCs), we found that Plexin-B2 knockout (KO) resulted not only in reduced tumor spread, but also strikingly a change of migration preference from axon tracts to peri-vascular routes. Moreover, while GSCs spread on both soft and stiff substrates in stripe assays, Plexin-B2 KO GSCs congregated on stiff stripes, a migratory behavior known as durotaxis. The ability of Plexin-B2 to empower GBM cells to overcome durotaxis tendency has clinical significance: as GBM progresses, the bulk of the tumor gradually stiffens, in part from increased pressure; tumor cells therefore must find a way to break from tumor bulk to invade softer brain parenchyma, and our preliminary data suggest that Plexin upregulation might fulfill this role. To further dissect mechanotension-induced GBM invasion, we will study mechanistic details of Plexin- B2 and -D1 as mechanoregulators in governing GBM invasion, with the aim to identify novel targets to curb GBM infiltration. In Aim 1, we will expand in vivo transplant studies to test how Plexin-mediated GBM invasion patterns and migratory paths applies to different GBM subtypes. We will then investigate how differentiation status and metabolic niche (hypoxia) may alter the choice of migration routes in dependence of Plexins. We will validate these findings in human GBM tissues. In Aim 2, we will delve into how Plexins operate to promote invasiveness. We will apply a series of mechanosensitive assays to interrogate Plexin-mediated biomechanical properties of migrating GBM cells, including intercellular adhesiveness, cell dispersion capacity, actomyosin dynamics, as well as infiltrative behavior in 3D vascular models. We will test how migrating GBM cells respond to different substrate stiffness, matrix substrates, and dissociated state, and how Plexins may alter durotactic behavior. We will define mechanoresponse pathways in GBM cells and use them as readouts to directly test Semaphorin- and mechano-dependent functions of Plexins. Finally, in Aim 3, we will interrogate downstream effectors of Plexin-B2 mechanosignaling. This includes the interaction of Plexins with the mechanosensitive Hippo/YAP pathway, and potential relay mechanisms through intracellular proteins Rap2 or AMOT. In sum, by studying mechano-sensitive mechanisms of GBM invasion, we explore new paradigms of GBM malignancy, with the ultimate goal to identify new therapeutic opportunities against this lethal cancer.