Establishment of 3D Tumor Organoids

3D organoid models were established as previously reported [32 (link)]. Briefly, approximately 1 cm³ of fresh tumor tissue was cut, soaked in PBS containing 5% penicillin–streptomycin and other antibiotics (2% Primocin, Invitrogen) for 30 min, and then minced. Enzymolysis of minced tissues was performed with 1.5 mg/mL collagenase IV (Catalog No. 7426, STEMCELL, British Columbia, Canada) for 1 h at 37 °C. Cells were washed twice with advanced DMEM/F12 and filtered through a 40-µm cell strainer (Catalog No. 27305, STEMCELL). After centrifugation at 1000 rpm for 5 min, the cells were inoculated into Matrigel (Catalog No. 356235, Corning, New York, USA) and overlaid with a human IntestiCult™ organoid growth medium (Catalog No. 06010, STEMCELL) containing 10 µM Y-27632, 1% Primocin and 1% penicillin–streptomycin. TrypLE™ Express Enzyme (Catalog No. 12604021, Thermo Scientific) was used to digest and resuspend the cells for organoid passaging.