Stiffness-dependent MSC homing and their differentiation to CAFs: Implications in breast cancer invasion.

Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here we examined how stiffness-dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment (TME) regulates cancer invasion. By analyzing previously published single-cell RNAseq datasets, we establish the existence of a sub-population co-expressing MSC and cancer associated fibroblast (CAF) markers in highly aggressive triple-negative breast cancers (TNBCs) in primary tumor, secondary sites, and in circulatory tumor cell clusters. Using 0.5, 2 and 5 kPa hydrogels mimicking different stages of ECM stiffening, we show that on pre-metastatic stroma mimetic 2 kPa gels, conditioned media secreted by MDA-MB-231 TNBC cells drives efficient MSC chemotaxis and induces stable CAF differentiation in a TGFβ/contractility-dependent manner. In addition to enhancing cancer cell proliferation, 2 kPa CAFs maximally boost local invasion and confer resistance to flow-induced shear stresses. Collectively, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBCs.