CAF contractility controls collagen stiffness through history-dependent competition between mechanical and proteolytic remodeling

Mechanical remodeling by cancer-associated fibroblasts (CAFs) stiffens tumors and can restrict molecular transport, yet the time-resolved coupling between cellular force and matrix mechanical state is poorly defined. Here we use a microfabricated force sensor integrated with 3D CAF-collagen tissue to measure CAF-generated force and collagen stiffness continuously during pharmacological perturbation. ROCK or non-muscle myosin II inhibition reduced force and remarkably drove collagen stiffness below its initial value rather than merely arresting stiffening. This softening was prevented by broad-spectrum MMP inhibition, but only modestly altered by lysyl oxidase inhibition, supporting a competition between force-driven collagen compaction and proteolytic remodeling. Second-harmonic generation imaging showed shorter, thinner and less aligned fibers after ROCK inhibition, while fluorescence recovery after photobleaching indicated faster dextran transport. Moreover, CAF contractile adaptation depended strongly on the duration of inhibition, with reversible recovery after brief treatment, a hyper-contractile rebound following prolonged treatment and washout, and partial force recovery during continuous exposure. These findings identify CAF-matrix mechanics as a dynamic, treatment-history-dependent balance and provide a framework for designing stromal interventions that alter stiffness and transport.