Plexin-B2 Function in Glioma Invasion

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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.