Matrix Stiffness Modulates 3D Spheroid-Derived Extracellular Vesicle Profiles and Discovery of Piezo1 Cargo.

Background: Increased extracellular matrix stiffness is a defining mechanical feature of solid tumors, yet how it regulates extracellular vesicle-mediated intercellular communication remains poorly understood in three-dimensional tumor microenvironments. Here, we demonstrate that ECM stiffness mechanistically regulates extracellular vesicle (EV) cargo loading in oral squamous cell carcinoma spheroids. Methods and Results: Using a tunable three-dimensional spheroid culture platform, we show that increased matrix stiffness enriches tumorigenic and metastatic non-coding RNA transcripts in EVs.  At a functional level, stiffness-primed EVs influence recipient spheroid growth by modulating proliferation and apoptosis. Notably, our study reveals that EVs are enriched in parental biomolecular cargo, including the mechanosensitive Piezo1 ion channel and adhesion and stemness molecule CD44. Protein expression and small RNA sequencing analyses confirm the incorporation of these components into spheroid-derived EVs in a stiffness-independent manner. Conclusion: Together, our findings identify ECM stiffness as a mechanistic regulator of EV composition and establish EVs as biomechanical signaling vectors that further influence cell proliferation in three-dimensional microenvironments. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s12195-026-00931-z.