Biophysical Regulation of Astrocytoma Cell Physiology in 2D and 3D Culture

The rapid progression of high-grade brain tumors is related to diffuse infiltration of single tumor cells into the surrounding brain parenchyma, a process that involves aberrant interactions between tumor cells and the extracellular matrix (ECM). Here, we show that biophysical cues from the ECM regulate key tumor cell properties relevant to invasion in both two-dimensional (2D) and three-dimensional (3D) culture models. We first investigated the role of ECM rigidity in regulating the structure, migration, and proliferation of a panel of astrocytoma cell lines on 2D fibronectin-coated polymeric ECM substrates of defined mechanical rigidity. On highly compliant ECMs, tumor cells appear rounded and fail to productively migrate. As ECM rigidity is increased, tumor cells spread extensively, form prominent stress fibers and mature focal adhesions, and migrate rapidly. Remarkably, cell proliferation is greatly enhanced on rigid versus compliant ECMs. Pharmacological inhibition of nonmuscle myosin II-based contractility blunts this rigidity-sensitivity and rescues motility on highly compliant substrates. We next explore astrocytoma mechanosensitivity in 3D by introducing a novel biomaterial platform in which we progressively modulate the biophysical properties of collagen I matrices by adding agarose. We find that agarose increases the bulk elasticity of 3D collagen ECMs over two orders of magnitude by forming a dense meshwork that intercalates between the entangled collagen fibers. Embedded glioma cells exhibit a pronounced transition to amoeboid motility accompanied by severe limitation of cellular invasion from multicellular spheroids as the agarose content of the hydrogels increases from 0-1% w/v. Our results are consistent with a model in which agarose structurally couples and reinforces individual collagen fibers, simultaneously introducing steric barriers to cell motility while shifting ECM dissipation of cell-induced stresses from the non-affine deformation of individual collagen fibers to the bulk-affine deformation of a continuum network.