Recombinant human fibroblast growth factor

SW620 and HCT116 cells were cultured in DMEM with 10% FBS in an incubator containing 5% CO₂ at 37°C. When cells reached 80% confluence, 6×10⁵ cells were resuspended in stem cell culture medium supplemented with 2% B27 (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml recombinant human epidermal growth factor (Gibco; Thermo Fisher Scientific, Inc.) and 20 ng/ml recombinant human fibroblast growth factor (Gibco; Thermo Fisher Scientific, Inc.) in each 6-well ultralow adhesion plates (Corning Inc.) for 6 days at 37°C in a 5% CO₂ atmosphere. When the tumorspheres grew to 50 µm in diameter, the spheres were imaged and counted under a light microscope (Nikon Corporation). The primary spheres were dissociated into single cells and cultured in stem cell culture medium (Gibco; Thermo Fisher Scientific, Inc.) for 14 days at 37°C to allow each cell forming one tumorsphere.

CTC-derived cells were maintained under hypoxic conditions (5% oxygen) on ultra-low attachment (ULA) 6-well plates (Corning, Cat# 3471-COR). CTC growth medium containing 20 ng/ml recombinant human Epidermal Growth Factor (GIBCO, Cat# PHG0313), 20 ng/ml recombinant human Fibroblast Growth Factor (GIBCO, Cat#100-18B), 1x B27 supplement (Invitrogen, Cat#17504-044) and 1x Antibiotic-Antimycotic (Invitrogen, Cat# 15240062) in RPMI 1640 Medium (Invitrogen, Cat# 52400-025) was added every third day. For passaging, cells were spun down at 800 g for 5 min using a Heraeus Multifuge X3R centrifuge (Invitrogen, Cat#75004515). The supernatant was subsequently aspirated and cells were resuspended in 2 ml/well CTC medium and plated in 6-well ULA plates. LM2 cells were passaged in DMEM/F-12 medium (Invitrogen, Cat#11330057) supplemented with 10% FBS (Invitrogen, Cat# 10500064) and Antibiotic-Antimycotic (Invitrogen, Cat# 15240062). For passaging, LM2 cells were washed once with D-PBS (Invitrogen, Cat#14190169) and dissociated using 0.25% Trypsin (Invitrogen, Cat#25200056).