Novel Mechanisms of Confined Migration of GBM Cells

SUMMARY Glioblastoma (GBM), the most common and lethal brain cancer is notorious for wide dissemination in the brain. Understanding cellular and molecular underpinnings of GBM invasion is thus critical. While earlier research has made progress on intrinsic drivers of cell motility, how GBM cells meet the challenge of extrinsic factors such as physical constraints to negotiate narrow paths through the brain parenchyma remains unclear. Our preliminary studies have revealed that endocytosis at the front of migrating cells is a critical process for membrane dynamics during confined migration. Furthermore, our new data have also implicated alteration of the negative electric charge at the inner membrane surface as a key organizer of cytoskeletal activity of migrating GBM cells in confined space. These novel preliminary findings were made possible by applying new microchannel devices coupled with live-cell imaging and advanced fluorescent probes to study GBM confined migration. In this exploratory R21 proposal, we will conduct mechanistic studies to test the hypothesis that membrane dynamics and membrane surface charge via mechanoelectric coupling are key drivers of confined GBM migration. In Aim 1, we will investigate how membrane dynamics mediated by endocytosis facilitates GBM confined migration. We will conduct functional assays to assess how specific endocytosis inhibitors impact membrane dynamics and migratory capacity of invading GBM cells through microchannels. In parallel, we will perform in vivo GBM transplants for proof-of-principle efficacy studies of limiting endocytosis to curb invasive spread of GBM. In Aim 2, we will explore the emerging concept of surface membrane charge and mechanoelectrical coupling for confined GBM migration. We will test the hypothesis that change of electrical charge at the inner leaflet of plasma membrane (by anionic lipids or membrane-associated proteins) bestows GBM cells with increased migratory capacity through organization of cytoskeletal activity. We will first generate novel molecular reagents to either increase or decrease surface charge in an inducible manner and test the impact on GBM confined migration. We will apply the microchannel devices for high-resolution live-cell imaging to monitor the effects of altering membrane charges on actin network, calcium flux, and endocytosis-mediated plasma membrane dynamics. We will then conduct proof-of concept in vivo GBM transplant studies to examine the efficacy of these novel molecular agents to impede GBM invasion. Together, our studies will provide mechanistic insight and conceptual advance on membrane plasticity and mechanoelectrical membrane dynamics for confined migration of GBM cells and their impact on GBM invasiveness in vivo. It will open translational opportunities to impede GBM spread and recurrence. We will also make the innovative microchannel devices available for the wider research community, thus accelerating the research of cancer mechanobiology and tumor invasion.