ECM stiffness-regulated exosomal thrombospondin-1 promotes tunneling nanotubes-based cellular networking in breast cancer cells.

Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging that may be further fine-tuned by the microenvironmental changes. Extracellular matrix (ECM) stiffening due to excess deposition and crosslinking of collagen is one of the crucial tumor-microenvironmental changes that influence a plethora of cellular processes, including cell-cell communication. We herein studied the crosstalk between exosomes and tunneling nanotubes (TNT), the two distinct means of cell-cell communication under varying ECM-stiffness conditions. We show that exosomes promote the formation of tunneling nanotubes in breast cancer cells, which results in cellular internet. Interestingly, exosomes drastically increased the fraction of cells connected by TNT; however, they elicited no effect on the number of TNTs per pair of connected cells or the length of TNT. The observed pro-TNT effects of exosomes were found to be ECM-stiffness dependent. ECM-stiffness tuned exosomes were found to promote TNT formation predominantly via the 'cell dislodgment model'. At the molecular level, exosomal thrombospondin-1 was identified as a critical pro-TNT factor. These findings underline the influence of ECM stiffening on two diverse modes of cell communication and their interdependence, which may have significant implications in cancer biomedical research.

ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1.

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.