Establishment of Colorectal Cancer Organoids

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.