A83 01

hTSCs were cultured as previously described (Okae et al., 2018 (link)). Briefly, a 6-well plate was coated with 5 μg/mL Collagen IV (Corning, 354233) at 37°C overnight. Cells were cultured in 2 mL TS medium [DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.2% FBS, 0.5% Penicillin-Streptomycin, 0.3% BSA, 1% ITS-X (Gibco, 51500), 1.5 μg/ml L-ascorbic acid (Wako, 013–12061), 50 ng/ml EGF (Rockland, 009–001 C26), 2 μM CHIR99021 (Stemgent, 04–0004), 0.5 μM A83-01 (BioVision, 1725), 1 μM SB431542 (BioVision, 1674), 0.8 mM VPA (Tocris, 2815), and 5 μM Y-27632] and in 5% CO₂ and 20% O₂. Media were changed every 2 days, and cells were passaged using TrypLE Express every 3 days at a ratio of 1:4. Unless otherwise specified, hTSCs between passage 10 and 20 were used for experiments.

The tumor organoids were isolated as previously described [10 (link)]. Briefly, cancer tissues were incubated with collagenase type II (Sigma-Aldrich, Louis, MO, USA), dispase type II (Roche Applied Science, Mannheim, Germany), and Y-27632 (BioVision, Mountain View, CA, USA) for 1 h at 37°C. Isolated cells were washed with PBS and centrifuged at 300 ×g for 3 min. The cells were then embedded in Matrigel (growth factor reduced, phenol red free; Corning, NY, USA) and seeded in 4-well plates, followed by the addition of the culture medium. The composition of the CRC organoid culture medium was 1 × B27 (Gibco, Grand Island, NY, USA), 1.25 mM N-acetyl cysteine (United States Pharmacopeia, Rockville, MD, USA), 50 ng/mL human epidermal growth factor (BioVision), 50 ng/mL human Noggin (Peprotech, Rocky Hill, NJ, USA), 10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (BioVision), and 100 mg/mL primocin (InvivoGen, San Diego, CA, USA). To prevent anoikis, 10 μM Y-27632 was added to the culture medium during the first 2-3 days. When organoids were >200 µm, they were passaged by pipetting using the Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's instructions.

hTSCs were cultured as previously described^{8 (link),10} . Prior to seeding, 6-well plates were coated with 5 μg/mL Collagen IV (Corning, 354233) at 37 °C overnight. Cells were cultured in 2 mL hTSC medium [DMEM/F12 (Gibco, 11320) supplemented with 0.1 mM 2-mercaptoethanol (Millipore Sigma, 8.05740), 0.2% FBS (Millipore Sigma, ES-009-B), 0.5% Penicillin-Streptomycin (Gibco, 15140), 0.3% BSA (Gibco, 15260), 1% ITS-X (Gibco, 51500), 1.5 μg/ml L-ascorbic acid (Wako, 013-12061), 50 ng/ml EGF (Peprotech, AF-100-15), 2 μM CHIR99021 (Stemgent, 04-0004), 0.5 μM A83-01 (BioVision, 1725), 1 μM SB431542 (BioVision, 1674), 0.8 mM VPA (Sigma, P4543), and 5 μM Y-27632 (Stemgent, 04-0012)] and in 5% CO₂ and 20% O₂. Tissue culture media were filtered using a 0.22 μm filter. Media were changed every 2 days, and cells were passaged using TrypLE Express (Gibco, 12604). Unless otherwise specified, hTSCs within 30 passages were used for experiments. The cell culture is regularly tested and confirmed negative for mycoplasma contamination.