3D Nanofibrillar Matrix Stiffness Modulates Extracellular Vesicle Cargo and Pro-Tumour Functions.

Extracellular matrix (ECM) stiffness and extracellular vesicles (EVs) are critical regulators of tumour progression, yet their interaction in three-dimensional (3D) microenvironments remains poorly understood. Most studies on ECM stiffness and EV biology rely on 2D cultures, which do not capture the complexity of the tumour microenvironment. Here, a biomimetic 3D nanofibrillar ECM model based on a cellulose nanofibril hydrogel was established to assess stiffness-dependent changes in EV properties and functions. EVs derived from stiff matrices (StEVs) exhibited distinct physicochemical characteristics and carried unique protein and microRNA cargo compared with those from soft matrices (SoEVs). Functionally, StEVs more potently promoted tumour cell proliferation and migration, while in vivo mouse models further demonstrated that StEVs enhanced tumour growth. Multi-omics analyses and pharmacological inhibition studies revealed that StEVs activate the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK1/2) signalling pathway in recipient cells. These findings highlight the mechanobiological regulation of EV-mediated intercellular communication within 3D ECM environments and demonstrate how matrix stiffness shapes EV cargo and pro-tumour activity.