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). Tremendous effort has been devoted to elucidating the genetic and biochemical underpinnings of these tumors; however, poor translation of candidate therapies from animal models to human patients has only increased the sense of urgency for the development of new approaches in both the laboratory and the clinic. Indeed, despite decades of extensive clinical and biological research, the life expectancy of patients with grade IV glioblastoma multiforme (GBM) brain tumors is still approximately one year at diagnosis. The work presented in this dissertation has approached this problem from a biophysical perspective, demonstrating that biomechanical cues from the ECM serve as regulators of key GBM 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 glioma cell lines on 2D fibronectin-coated polymeric ECM substrates of defined mechanical rigidity. We found that 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, providing support for a novel model in which ECM rigidity provides a transformative, microenvironmental cue that acts through actomyosin contractility to regulate the invasive properties of GBM tumor cells.
We next explored glioma 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 found 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 from mesenchymal to amoeboid modes of motility, accompanied by severe limitation of cellular invasion from multicellular spheroids as the agarose content of the hydrogels increases from 0-1% w/v. Furthermore, addition of agarose progressively inhibits cell-directed macroscopic gel compaction, concomitant with restriction of cell spreading and reduced cytoskeletal stress fiber and focal adhesion assembly. While time-of-flight secondary ion mass spectrometry and scanning electron microscopy fail to reveal agarose-induced alterations in collagen ligand presentation, the latter modality shows that agarose strongly impairs cell-directed assembly of large collagen bundles. Our results support 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.
Additionally, we found that agarose-mediated inhibition of cell spreading and cytoarchitecture on 3D collagen-agarose composite hydrogels can be rescued by partial beta-agarase digestion of the agarose network or by covalently crosslinking the matrix with glutaraldehyde. Based on these results, we argue that cell spreading and motility on collagen requires local matrix stiffening, which can be achieved via cell-mediated fiber remodeling or by chemically crosslinking the fibers. These findings underscore the critical role of ECM mechanics in regulating glioma tumor cell behaviors in 3D matrices, and bear general implications for micromechanical regulation of cell adhesion and motility in composite fibrillar-nonfibrillar ECMs in vivo and the use of such matrices in tissue engineering and regenerative medicine applications.
