Substrate Stiffness Modulates Fibroblast Extracellular Vesicle Secretion Via Mechanotransduction Pathways

Abstract The extracellular matrix (ECM) is recognized as a key regulator of cell behavior, with its stiffness playing a crucial role in the progression of pathological conditions such as cancer and cardiovascular diseases. While extracellular vesicles (EVs) are essential mediators of intercellular communication, the influence of matrix stiffness on EV secretion remains poorly understood. This study investigates how substrate stiffness affects EV size and composition in mouse mammary and cardiac fibroblasts, the key stromal cell types in breast cancer and cardiac microenvironments. Importantly, stiffness‐tuned EV proteomic cargo is uncovered, providing new insights into how mechanical cues can reprogram the signaling functions of fibroblast‐derived vesicles. The findings show that substrate stiffness significantly alters EV characteristics, with sizes increasing below stiffnesses of 20 kPa and decreasing on stiffer substrates. Mechanotransduction pathways involving p53 and thioredoxin are identified as regulators of these alterations, with thioredoxin dominate the modulation in mammary fibroblasts and p53 in cardiac fibroblasts. These results underscore the importance of ECM stiffness in modulating EV secretion and highlight candidate pathways influenced by ECM remodeling that may warrant further investigation for therapeutic relevance.