Updated publication reference for PubMed record(s): 31160380. The ability of primary tumour cells to invade and metastasise is responsible for over 90% of cancer patient deaths. During tumour growth and progression, cancer cells secrete factors that recruit and reprogram otherwise healthy cells to facilitate the formation of a favourable tumour microenvironment. Here, we have investigated how breast cancer cells convert normal mesenchymal stromal cells (MSCs) into tumour-associated MSCs (TA-MSCs) using unbiased global approaches. We compared the secretomes produced by non-invasive MCF-7 cells with invasive MDA-MB-231 cells, and identified extracellular matrix and exosome components associated with invasion. We then treated MSCs cultured in fully-defined, synthetic 3D hydrogels with invasive/non-invasive cancer secretomes and analysed their responses by kinase activity profiling and RNA sequencing, which led us to identify an invasion-associated signature of MSC conversion. Finally, we investigated whether there is an organ-specific metastasis-associated reprogramming of MSCs, and found that breast cancer cells from different metastatic sites have different secretome profiles that induce reprogramming of MSCs. These data describe at a systems-level how breast cancer cells with different invasion and metastatic abilities secrete molecules that activate MSCs and convert them into TA-MSCs.