(723c) Mechanisms of Three-Dimensional Glioma Cell Motility in Non-Fibrillar Matrices

Diffuse infiltration of single cells into brain parenchyma is a hallmark of malignant glioma. This extreme invasiveness makes complete tumor resection difficult and contributes to the high mortality rate associated with this disease. Brain parenchyma has a distinct physical structure characterized by densely packed neural cell processes and sub-micron extracellular space, and is largely devoid of the fibrillar collagen scaffolding typically found in stromal tissue. Consequently, glioma cells migrating in brain slices exhibit a distinct type of motility, with branched protrusions and hourglass-shaped cell-body deformations that help squeeze cells through tight spaces (Beadle et al., Mol. Biol. Cell 2008; 19(8):3357-68). However, the mechanistic details of this unique mode of motility remain incompletely understood. To address this question, we synthesized brain-mimetic nanoporous, non-fibrillar extracellular matrices (ECMs) based on cross-linked hyaluronic acid (HA), the major component of brain ECM, and functionalized them with Arg-Gly-Asp (RGD)-containing peptides to facilitate integrin-mediated cell adhesion. In a three-dimensional (3D) spheroid invasion paradigm, glioma cell motility was highly reminiscent of that seen in brain slices, validating the use of these ECMs a model system (Ananthanarayanan et al. Biomaterials 2011; 32(31):7913-23). Eliminating RGD peptides from the ECM abolished invasion, suggesting a requirement for integrin-mediated adhesions for this mode of motility. Increasing matrix density, or inhibiting myosin-based cellular contractility by blebbistatin or shRNA-induced knockdown of Myosin IIA also severely impaired motility. We also report preliminary results from two sets of studies: First, we investigate the balance of protrusive and contractile forces in glioma cell motility by pharmacologically and genetically manipulating the Rho GTPases RhoA, Rac1, and Cdc42. Second, we explore the involvement of actin-nucleating proteins such as Arp2/3 and formins in the protrusive dynamics observed in this mode of motility. Our studies collectively help define the mechanotransductive signaling mechanisms that underlie the distinct mode of glioma cell motility observed in non-fibrillar matrices.