Plexin-B2 Function in Glioma Invasion

恶性脑肿瘤胶质母细胞瘤（GBM）是高度浸润性的。迁移的GBM细胞暴露于 然而，在肿瘤侵袭过程中的生物力学力，对机械敏感性通路知之甚少 使GBM细胞获得侵袭力。在这里，我们假设丛状蛋白指导受体，集中在这一点， 关于丛蛋白-B2及其副产物丛蛋白-D1的建议（两者在GBM中高度上调，并且与GBM中的低表达相关） 患者存活率），可能作为关键的机械调节剂，以增强机械张力诱导的胶质瘤细胞 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（GSC），我们发现丛蛋白-B2敲除（KO）不仅导致 减少肿瘤扩散，但也显著改变了从轴突束到血管周围的迁移偏好 航线此外，虽然GSC在条纹测定中在软基质和硬基质两者上扩散，但丛状蛋白-B2 KO GSC 聚集在坚硬的条纹上，这是一种叫做硬脊膜扩张的迁移行为。Plexin-B2能够增强 GBM细胞克服硬脊膜倾向具有临床意义：随着GBM进展，肿瘤体积增大， 逐渐变硬，部分原因是压力增加;因此，肿瘤细胞必须找到一种方法来打破肿瘤 大量侵入较软的脑实质，我们的初步数据表明，丛状蛋白上调可能实现 这个角色。为了进一步剖析机械张力诱导的GBM侵袭，我们将研究丛状蛋白的机制细节， B2和-D1作为控制GBM侵袭的机械调节剂，目的是确定抑制GBM侵袭的新靶标。 GBM渗透。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。然后我们将研究如何区分 状态和代谢生态位（缺氧）可能改变依赖丛蛋白的迁移途径的选择。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Plexins如何运作，以促进 侵略性我们将应用一系列的机械敏感性测定来询问丛蛋白介导的生物力学。 迁移性GBM细胞的特性，包括细胞间粘附、细胞分散能力、肌动球蛋白 动力学，以及3D血管模型中的浸润行为。我们将测试迁移的GBM细胞如何响应 不同的基质硬度，基质基质和解离状态，以及丛状蛋白如何改变硬规立构 行为我们将定义GBM细胞中的机械反应途径，并将其用作直接测试的读数。 神经丛蛋白的脑信号蛋白和机械依赖性功能。最后，在目标3中，我们将询问下游 丛蛋白-B2机械信号的效应子。这包括丛状蛋白与机械敏感性 Hippo/雅普通路，以及通过细胞内蛋白Rap 2或AMOT的潜在中继机制。总之，由 通过研究GBM侵袭的机械敏感性机制，我们探索了GBM恶性肿瘤的新范例， 最终目标是找到针对这种致命癌症的新治疗方法。

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.