Matrix Stiffness and Solid Stress Modulate 3D Hydrogel Growth via Yes-associated Protein Signaling in Neurofibromatosis Type 2

Introduction: : NF2-associated schwannomatosis (NF2-SWN) is a devastating genetic tumor predisposition syndrome whose hallmark is bilateral vestibular schwannomas (VS) which cause deafness and brainstem compression. There are no FDA-approved pharmacotherapies for NF2, and the current drug discovery pipeline relies on cell lines cultured on tissue culture plastic. VS tumorigenesis occurs within bony confinement of the internal auditory canal under mechanical compression and schwannoma cells experience significant alterations in its mechanical microenvironment. Accumulation of solid stress within the tumor influences disease progression in cancer. However, the degree to which extracellular matrix stiffness contributes to the accumulation of solid stress in NF2 schwannoma has not been investigated. Here, we generate 3D schwannoma culture in a confining environment using agarose and collagen hydrogels. We characterize how matrix stiffness and type of biomaterials affect the phenotype of schwannoma cells.

Materials and

Methods: : Human NF2 schwannoma (HEI-193) cells and primary VS from surgical specimens were embedded in hydrogels comprised of either low melting-temperature agarose (0.35%-4%) or collagen (2-8 mg/mL). Additionally, cells were cultured on 2D collagen (2 mg/mL) or collagen - agarose (1-4%) composite gels. Storage modulus was quantified by rheology. Brightfield images were used to quantify cell morphology. Gels were stained with acridine orange and propidium iodide to quantify live and dead cells. Immunofluorescence staining was performed using N-cadherin, YAP, MMP9, and pFAK. The topology of collagen was quantified using picrosirius red staining. The expression of genes involved in mechanotransduction (TRPV4, PIEZO1, FAK) and mature Schwann cell markers (OCT6, SOX10) was determined by qRT-PCR.

Results, Conclusions, and Discussions:: HEI-193 cells were readily encapsulated in 3D hydrogels with excellent viability (>85%) in a wide range of storage moduli (19 Pa to 7 kPa) (Fig. 1A-B). As agarose concentration increased, there was a reduction in cell proliferation over 7 days (Fig. 1C). We examined whether stiffer hydrogels led to alterations in cell/cell and cell/matrix interactions and YAP signaling. Cellular spheroid area decreased by over 50%. There was a significant decrease in N-cadherin with increased agarose concentrations. Nuclear-to-cytoplasmic YAP expression ratio was significantly upregulated in stiffer hydrogels. Primary VS cells showed similar trends, including decrease cell area, reduced N-cadherin expression, and increased nuclear-to-cytoplasmic YAP ratio (Fig. 1D). To determine if hydrogel stiffness governs mechanical stress and whether schwannoma cells retain “mechanical memory”, we partially degraded agarose gels to induce stress relief (storage modulus reduction by >90%) (Fig. 1E). There was a corresponding increase in cell area as well as normalization of N-cadherin and YAP expression to pre-stress relief levels, suggestion that stiff hydrogels imposed confining stress on schwannoma cells without significantly retained mechanical memory (Fig. 1F).
Since agarose is inert, we investigated whether schwannoma cells behaved similarly within a biologically active collagen/agarose hydrogel. In 2D collagen-agarose composite gels, increased agarose activated FAK YAP and MMP9. TRPV4 and FAK expression were upregulated by over 10-fold (Fig. 2A-B). We also examined the cell phenotype in 2D versus 3D collagen hydrogels. Increased collagen concentration increased hydrogel stiffness, which resulted in a more elongated cell morphology, and increased mature Schwann cell marker expression (Fig. 2C-D). Ongoing work is focusing on quantitively assessing collagen network topology and architecture using an automated computational platform, to determine whether increased fiber disorganization from cell-mediated remodeling is occurring in stiff hydrogels (Fig. 2E).
Our results demonstrate that schwannoma cells dynamically respond to extrinsic solid stress. This study highlights the critical importance of modeling dynamic alterations in the mechanical environment during tumor progression in NF2-associated VS. The novel 3D tumor hydrogel model offers excellent translational potential for understanding mechanotransductive pathways and enabling therapeutic testing. Finally, this platform is tunable to different cell types in other neurofibromatosis and other matrix modifications.

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