Decoupling Mechanical Confinement and Fibrotic Extracellular Matrix Signaling in Vestibular Schwannoma Using Tunable 3D Hydrogels

Abstract Purpose Vestibular schwannoma (VS) progressively stiffens and remodels its extracellular matrix (ECM) during growth. However, how mechanical confinement and adhesive ECM signaling regulate schwannoma behavior in vitro remain incompletely defined. Methods Human Nf2 -/- schwannoma and primary VS cells established from fresh surgical specimens were cultured in complementary 3D hydrogel platforms. Biochemically inert agarose hydrogels spanning physiologic to pathologic stiffnesses created a non-adhesive mechanical confinement environment, while type I collagen hydrogels modeled an adhesive environment with a dense, fibrillar matrix characteristic of fibrotic tumor ECM. Hydrogel stiffness was quantified by rheology. Cell viability, proliferation, morphology, mechanosensitive signaling, and ECM remodeling were quantified. Mechanical stress was relieved by enzymatic degradation. YAP activity was pharmacologically inhibited, and transforming growth factor-β (TGF-β) was used to induce collagen remodeling. Results Under non-adhesive confinement, increasing stiffness suppressed schwannoma reduced cell spreading, decreased N-cadherin expression, and increased nuclear YAP localization. Stress relief reversed YAP activation while enabling enhanced proliferative recovery and increased N-cadherin–associated adhesion. Cells under increased confinement exhibited increased sensitivity to YAP inhibition, indicating confinement-dependent reliance on mechanotransduction. In contrast, adhesive ECM conditions supported active matrix remodeling with increasing stiffness, including elevated activities of MMP9 and phosphorylated focal adhesion kinase (pFAK). TGF-β induced both collagen disorganization and SMAD3 nuclear localization, which was attenuated by YAP inhibition. Conclusions Mechanical confinement and ECM composition drive distinct, context-dependent adaptation programs in VS. As stiffness increases, cells in non-adhesive environments adopt a reversible, YAP-associated stress response, while an adhesive ECM shifts behavior toward matrix remodeling and cell adhesion-driven signaling.