Colorectal Tumor-Derived Organoid Culture

Human colorectal tumor tissue-derived ALI organoids were cultured as described previously [10 (link)]. The medium components were as follows: Advanced Dulbecco’s Modified Eagle’s Medium (DMEM) with 50% Wnt, Noggin and R-Spondin conditioned medium; GlutaMax; B-27 supplement; 100 μg/mL Primocin (Invitrogen, Carlsbad, CA, USA); 1 mM N-Acetyl-l-cysteine; 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA); 50 ng/mL mouse epidermal growth factor (EGF) (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); and 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Stem cell-related signal inhibitors were as follows: YO-01027 (Toronto Research Chemicals, Toronto, Canada); DAPT (Adooq Bioscience); WAV939; Wnt-C59; AY9944; and GANT61 (Cayman). Anti-cancer drugs were as follows: 5-FU (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA); and Oxaliplatin (Adooq Bioscience). Antibody sources were as follows: GLI-1 (Gene Tex, Irvine, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA); c-Myc; and Nanog (Cell Signaling, Beverly, MA, USA). Secondary antibodies were as follows: Horseradish peroxidase (HRP) conjugated anti-rabbit IgG; HRP conjugated anti-goat IgG (Cayman); and HRP conjugated anti-mouse IgG (Millipore, Temecula, CA, USA).