Penicillin

Human brain microvascular endothelial cells (HBMVECs) were purchased from Cell Systems (Kirkland, WA, USA) and grown on attachment factor-coated plates in CSC complete serum-free medium (Cell Systems, Seattle, WA, USA) or in M199 medium supplemented with 20% FBS, 5 U/mL of heparin, 3 ng/mL of recombinant human fibroblast growth factor-basic (FGF-β; Millipore, Temecula, CA, USA), penicillin (100 U/mL), and streptomycin (100 μg /mL) at 37 °C in an atmosphere of 5% CO₂ in air. Human leukemia-60 (HL-60) cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

CAFs were isolated as previously described.^{45 (link)} Briefly, tissue from early-stage IDC (stage 1) that was less than 10 mm in diameter was sliced and then digested overnight with a collagenase preparation (ISU ABXIS; Seoul, South Korea). Digested tissue was filtered through a 70 μm cell strainer (SPL Life Science; Pocheon-si, South Korea). Cells were separated by Ficoll gradients, washed with PBS, resuspended with DMEM/F12 cell culture medium containing 20% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL; Grand Island, NY, USA) and cultured at 37 °C in a humidified incubator containing 5% CO₂. The fibrotic nature of the isolated cells was confirmed by microscopic determination of morphology and immunofluorescence characterization using the antibodies against vimentin (Abcam; Cambridge, UK), cytokeratin (Dako; Glostrup, Denmark) and cytokeratin 5 (Novocastra; Newcastle upon Tyne, UK). Breast cancer cell lines (BT-474, MCF7, SK-BR-3, MDA-MB-231) were purchased from Korean Cell Line Bank (Seoul, South Korea) (authenticated using morphology and STR profiling) and cultured with DMEM cell culture medium containing 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator containing 5% CO₂.