Stiffness-dependent MSC homing and differentiation into CAFs - implications for breast cancer invasion

Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here, we examine how stiffness -dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment regulates cancer invasion. By analyzing previously published single -cell RNA sequencing datasets, we establish the existence of a subpopulation of cells in primary tumors, secondary sites and circulatory tumor cell clusters of highly aggressive triplenegative breast cancer (TNBC) that co -express MSC and cancerassociated fibroblast (CAF) markers. By using hydrogels with stiffnesses of 0.5, 2 and 5 kPa to mimic different stages of ECM stiffening, we show that conditioned medium from MDA-MB-231 TNBC cells cultured on 2 kPa gels, which mimic the pre -metastatic stroma, drives efficient MSC chemotaxis and induces stable differentiation of MSC -derived CAFs in a TGF0 (TGFB1)- and contractility -dependent manner. In addition to enhancing cancer cell proliferation, MSC -derived CAFs on 2 kPa gels maximally boost local invasion and confer resistance to flow -induced shear stresses. Collectively, our results suggest that homing of MSCs at the premetastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBC.