Generating CAF-like Cells from HMEC-1

CAF-like cells were obtained by TGF-β2 stimulation of human microvascular endothelial cells (HMEC-1) as described previously [35 (link),36 (link),37 (link)]. HMEC-1 were cultured in MCDB131 (Life Technologies, Paisley, UK) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) or with one of CM: co-CM1, co-CM2, co-CM1 + VIN, co-CM2 + VIN, CAFs-CM1, CAFs-CM2, CAFs-CM1 + VIN, CAFs-CM2 + VIN, or CAFs-CM. All lines were maintained at 37 °C in a humidified 5% CO₂ atmosphere. CM was recovered from colon cancer cells grown for 72 h. In some experiments, the cells were treated with vincristine (5 nM) or NSAIDs: 2500 µM aspirin (AsA) and 400 µM ibuprofen (IBU). The cells were harvested by 0.05% trypsin-EDTA and washed with phosphate-buffered saline (PBS).