*2.2. Cell Lines and uPA and uPAR Expression*

The human mammary epithelial invasive ductal carcinoma cell lines MCF-7 and MDA-MB-231 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) containing 24 mM NaHCO<sup>3</sup> and supplemented with 10% (*v*/*v*) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Cells were maintained in culture at 37 ◦C in a 95% humidified atmosphere with 5% CO<sup>2</sup> in a HERAcell incubator (Kendro Laboratory Products, Germany). For passaging, the cells were harvested by treatment with 0.05% trypsin-EDTA (Life Technologies, CA, USA), followed by centrifugation at 300× *g* for 5 min. For the experiments, the cells were harvested by treatment with PBS containing 5 mM EDTA (pH 7.4), followed by centrifugation at 300× *g* for 5 min. Viable cells were counted with a hemocytometer using the Trypan Blue (Sigma-Aldrich, MO, USA) exclusion method. Cell lines were routinely tested and confirmed to be negative for mycoplasma contamination (in-house testing conducted by the Illawarra Health and Medical Research Institute Technical Services Unit). Cell lines were confirmed to be negative for cross-contamination by short-tandem repeat (STR) sequencing (performed by the Garvan Institute of Medical Research, Darlinghurst, Australia).

Expression of uPA and uPAR on the surface of cells was determined by flow cytometry, as described in Supplementary Information.
