Maintaining and Differentiating Cancer Stem Cells

The cancer stem cells (CSCs) used in this study (PANC-1, PSN-1, SW620, HT29, WiDr, and SW480) were maintained as monolayer stem cell cultures [28 (link),29 (link)]. Briefly, the cells were cultured on collagen-I-coated dishes (IWAKI, Tokyo, Japan) in stem cell culture medium (DMEM/F-12 supplemented with 1% B27 (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL of EGF and FGF2 (Peprotech, Rocky Hill, NJ, USA), D-(+)-glucose (final concentration, 26.2 mM), L-glutamine (final concentration, 4.5 mM), 100 units/mL of penicillin, and 100 mg/mL of streptomycin). The stem cell culture medium was changed every 3 days, and EGF and FGF2 were added to the stem cell culture medium daily. To obtain isogenic non-CSC counterparts, the CSCs were induced to lose their stemness by culturing in DMEM/F-12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 units/mL of penicillin, and 100 mg/mL of streptomycin for 1 week. Then, the cells were used in the experiments in this study as non-CSCs.