Tissue dissociation and organoid culture

The organoid culture was performed as previously described [3], with minor modifications to the tissue dissociation protocol to improve cell viability. Specifically, well-washed tumor-adjacent normal tissues were carefully cut into 1–3-mm-long fragments by a scalpel. The muscle layer was then stripped off from the mucosa layer under a dissecting microscope. Subsequently, fragments were incubated in 5μM EDTA for 15 min with vigorously shaking. The isolated crypts were collected and further washed twice with the basal medium. Then, the crypts were allowed to mix with BME (Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Bio-techne) and seeded in 24-well plates (50 μL BME/well). Culture medium was added after the BME was well solidified (approximately 20 min after seeding). Tumor tissues were then cut into small pieces and digested by collagenase (type II and type IV; Invitrogen) for 30 min at 37 °C with vigorous pipetting every 5 min. After digestion, dissociated tissues were passed through a 40-μm cell strainer (Corning). The cell suspension was first centrifuged at 400 g for 5 min at 4 °C and then was re-suspended with BME and seeded in 24-well plates (50 μL BME/well). The composition of the organoid culture medium was as follows:

The chemical-defined medium are advanced DMEM/F12 (Gibco), 100 U/mL penicillin/streptomycin (Gibco), Primocin (InvivoGen), 2 mM GlutaMAX (Gibco), 0.5 μM A83-01 (Tocris), 1x B27 (Gibco), 5 μM SB202190 (Sigma), 100 nM prostaglandin E2 (Tocris), 0.5 μg/mL R-spondin (Peprotech), 4mM nicotinamide (Sigma), 10 nM gastrin I (Sigma), 50 ng/ml EGF (Peprotech), 100 ng/ml Noggin (Peprotech), 100 ng/mL WNT3A (Millipore), 10ng/ml FGF-10 (Peprotech), and 10ng/ml FGF-basic (Peprotech). Notably, 10 μM Y-27632(Selleck) was supplemented to the medium in the first week and the medium was changed every 2 days.

Conditioned medium are 50% conditioned media (1:1 diluted with Advanced DMEM/F12), 100 U/mL penicillin/streptomycin, Primocin (InvivoGen), 2 mM GlutaMAX (Gibco), 0.5 μM A83-01 (Tocris), 1x B27 (Gibco), 5 μM SB202190 (Sigma), 100 nM prostaglandin E2 (Tocris), 4mM nicotinamide (Sigma), 10 nM gastrin I (Sigma), 50 ng/mL EGF (Peprotech), 10ng/mL FGF-10 (Peprotech), and 10ng/ml FGF-basic (Peprotech). The conditioned medium was prepared according to the protocol as previously described [13].