Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform

Abstract

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by diffuse infiltration of single cells into the brain parenchyma, which is a process that relies in part on aberrant biochemical and biophysical interactions between tumor cells and the brain extracellular matrix (ECM). A major obstacle to understanding ECM regulation of GBM invasion is the absence of model matrix systems that recapitulate the distinct composition and physical structure of brain ECM while allowing independent control of adhesive ligand density, mechanics, and microstructure. To address this need, we synthesized brain-mimetic ECMs based on hyaluronic acid (HA) with a range of stiffnesses that encompasses normal and tumorigenic brain tissue and functionalized these materials with short Arg-Gly-Asp (RGD) peptides to facilitate cell adhesion. Scanning electron micrographs of the hydrogels revealed a dense, sheet-like microstructure with apparent nanoscale porosity similar to brain extracellular space. On flat hydrogel substrates, glioma cell spreading area and actin stress fiber assembly increased strongly with increasing density of RGD peptide. Increasing HA stiffness under constant RGD density produced similar trends and increased the speed of random motility. In a three-dimensional (3D) spheroid paradigm, glioma cells invaded HA hydrogels with morphological patterns distinct from those observed on flat surfaces or in 3D collagen-based ECMs but highly reminiscent of those seen in brain slices. This material system represents a brain-mimetic model ECM with tunable ligand density and stiffness amenable to investigations of the mechanobiological regulation of brain tumor progression.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…In the recent years, scientists have set up HA-based scaffolds in combination with other molecules, such as a different type of collagens [230,234], the kappaelastin (HA-E), present in the basement membrane of blood vessels [235], gelatine, PEG [231], and chitosan [232]. Besides these natural extracellular matrix materials, which are expensive and potentially could transmit pathogens [236], several natural polymers have been also studied, such as chitosan-alginate scaffold [125], or alginate hydrogel functionalized with D-or L-aminoacids [237].…”

“…Moreover, several other matrix compositions have been tested with the aim of better reproducing the in vivo microenvironment. Thus, invasion assays have been performed in HA-based hydrogel [230,[233][234][235]]. …”

Shooting for the moon: using tissue-mimetic hydrogels to gain new insight on cancer biology and screen therapeutics

“…[56] Ananthanarayanan et al used hydrogels comprising hyaluronic acid methacrylate functionalized with cysteine-containing RGD peptides and crosslinked with dithiothreitol in a Michael-type addition reaction to investigate the effects of matrix stiffness on glioblastoma phenotype. [60] They found that spheroids of glioblastoma cells displayed a more invasive phenotype marked by more infiltration into the surrounding matrix from the spheroid boundary in 3D culture in soft (150 Pa) gels compared with stiff gels. Importantly, this result was in contrast to 2D culture experiments on the hydrogels, in which the glioblastoma cells displayed more spread morphology, faster migration, and more proliferation on stiff gels (1 and 5 kPa), similar to what was observed by Ulrich et al [55] These conflicting responses to matrix stiffness in 2D and 3D models may indicate that the dimensionality of the culture has a significant impact on the cellular response.…”

Biomimetic Strategies for the Glioblastoma Microenvironment

“…Irrelevant components of the brain ECM, such as chitosan-alginate hydrogels (Kievit et al, 2010) and collagen-agarose hydrogels (Ulrich et al, 2010), were chosen because they have a biologically inert to investigate biophysical effects of the matrices solely, resulting in a mechanosensitive Umesh et al, 2014), have been performed to examine the role of mechanobiological regulation in GBM invasion. For investigating mechanosensing characteristics of GBM cells, the modified forms of HA gels, such as HA-methacrylate (Me-HA) or RGDfunctionalized Me-HA (Ananthanarayanan et al, 2011;Kim and Kumar, 2014), were used to control the matrix stiffness, observing the stiffness-dependent GBM features via CD44-mediated mechanosensing.…”

“…Therefore, to investigate the role of HA in GBM invasion, the HA is chemically modified so that it is cross-linked. Chemically modified forms of HA hydrogels include thiolated HA (Rao et al, 2013a), methacrylated HA (Ananthanarayanan et al, 2011;Kim and Kumar, 2014), and mixtures with GelMA . GBM cells are strongly influenced by the HA concentration and show increased invasion via CD44-mediated HA adhesion (Kim and Kumar, 2014).…”