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Breast cancer progression features ECM stiffening due to excess deposition and crosslinking of collagen, which dramatically influence tumor behaviour and fate. The mechanisms by which extracellular matrix (ECM) stiffening drives breast cancer invasion is an area of active research. Here we demonstrate the role of exosomes in ECM stiffness triggered breast cancer invasiveness. Using stiffness tuneable hydrogel ECM scaffolds, we show that stiff ECMs promote exosome secretion in a YAP/TAZ pathway-dependent manner. Interestingly, blocking exosome synthesis and secretion by GW4869 abrogated stiffness regulated motility and contractility in breast cancer cells. Reciprocally, exogenous addition of ECM stiffness-tuned exosomes orchestrated a series of changes in cell morphology, adhesion, protrusion dynamics resulting in fostered cell motility and invasion. Proteomic analysis of exosomal lysates followed by overrepresentation analysis and interactome studies revealed enrichment of cell adhesion and cell migration proteins in exosomes from stiff ECM cultures compared to that of soft ones. Quantitative proteomics of exosomes combined with genomic analysis of human breast tumor tissues (TCGA database) identified thrombospondin-1 (THBS1) as a prospective regulator of stiffness-dependent cancer invasion. Knockdown studies confirmed that the pro-invasive effects of stiffness-tuned exosomes are fuelled by exosomal THBS1. We further demonstrated that exosomal THBS1 mediates these stiffness-induced effects by engaging matrix metalloproteinase and focal adhesion kinase. Our studies establish the pivotal role of exosomal communication in ECM stiffness dependent cell migration with exosomal THBS1 as a master regulator of cancer invasion, which can be further exploited as a potential theranostic for improved breast cancer management.

Stem cell differentiation is dictated by the dynamic crosstalk between cells and their underlying extracellular matrix. While the importance of matrix degradation mediated by enzymes such as matrix metalloproteinases (MMPs) in the context of cancer invasion is well established, the role of MMPs in stem cell differentiation remains relatively unexplored. Here we address this question by assaying MMP expression and activity during differentiation of mouse embryonic stem cells (mESCs) on mouse embryonic fibroblast (MEF) derived matrices (MEFDMs) of varying stiffness and composition. We show that mESC differentiation into different germ layers is associated with expression of several MMPs including MMP-11, 2, 17, 25 and 9, with MMP-9 detected in cell secreted media. Different extents of softening of the different MEFDMs led to altered integrin expression, activated distinct mechanotransduction and metabolic pathways, and induced expression of germ layer-specific markers. Inhibition of MMP proteolytic activity by the broad spectrum MMP inhibitor GM6001 led to alterations in germ layer commitment of the differentiating mESCs. Together, our results illustrate the effect of MMPs in regulating mESC differentiation on engineered cell derived matrices and establish MEFDMs as suitable substrates for understanding molecular mechanisms regulating stem cell development and for regenerative medicine applications.

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.