Organoid Culture

Fresh tumor and paired adjacent normal colon tissues were kept in phosphate-buffered saline solution without calcium and magnesium (PBS WO, Sigma Aldrich) at 4°C. The isolation of healthy crypts and tumor epithelium was performed as described by Sato et al. (10). The organoid culture medium was refreshed every two days. Culture medium including 50% L-WRN condition medium (containing Wnt3a, R-Spondin1, and Noggin), 1X Glutamax, 1X hepes, 1X N2 (Invitrogen), 1X B27 minus vitamin A (Life Technologies), 1X penicillin/streptomycin solution (Invitrogen), 50 ng/mL human EGF (Gibco), 1 mM N-acetylcysteine (Sigma Aldrich), 10 mM nicotinamide (Sigma Aldrich), 10 nM gastrin (Sigma Aldrich), 10 µM SB202190 (Selleck), 0.01 uM PGE2 (Sigma Aldrich), and 10 mM Y27632 (Sigma Aldrich).