Extracellular Matrix Viscoelasticity Regulates Mammary Branching Morphogenesis

Structural and mechanical cues from the extracellular matrix (ECM) regulate tissue morphogenesis. Tissue development has conventionally been studied with ex vivo systems where the mechanical properties of the extracellular environment are either poorly controlled in space and time, lack tunability, or do not mimic ECM mechanics. For these reasons, it remains unknown how matrix stress relaxation rate, a time-dependent mechanical property that influences several cellular processes, regulates mammary branching morphogenesis. Here, the influence of matrix stress relaxation on mammary branching morphogenesis is systematically investigated using 3D alginate-collagen matrices and spheroids of human mammary epithelial cells. Slow stress relaxing matrices enhanced branching, which is accompanied by local collagen fiber alignment, intermittent pulling contractions applied to the ECM, and focal adhesion signaling. On the contrary, it is observed that growing spheroids in fast stress relaxing matrices applied isotropic pushing forces to the ECM. Pharmacological inhibition of both Rac1 and non-muscle myosin II prevented epithelial branch formation, regardless of matrix stress relaxation rate. Interestingly, restricting cellular expansion via increased osmotic pressure is sufficient to impede epithelial branching in slow stress relaxing matrices. This work highlights the importance of stress relaxation in regulating and directing mammary branch elongation.