Organoid Culture from Patient-Derived Xenografts

Organoids were established from PDX tissues as previously described^{23 (link),44 (link)}. Fresh or frozen-thawed single PDX cells were seeded in growth factor reduced, phenol red-free, ldEV-free Matrigel (Corning), with 1–1.5 × 10⁵ cells seeded per 20 µl of Matrigel in 48 well plates. Organoids were cultured in organoid media, consisting of advanced DMEM/F-12 media (Thermo Fisher) containing 1% penicillin-streptomycin, 2 mM Glutamax, 1 nM DHT, 1.25 mM N-acetylcysteine, 50 ng/ml EGF, 500 nM A83-01, 10 mM nicotinamide, 10 μM SB202190 (Sigma), 2% B27 (Life Technologies), 100 ng/ml noggin (Peprotech), 10 ng/ml FGF10 (VWR), 5 ng/ml FGF2, 1 μM prostaglandin E2 (Tocris), and 10% R-spondin 1 conditioned media. 10 μM Y-27632 dihydrochloride (Selleck Chemicals) was added to culture medium during organoid establishment and the first subsequent passage. To assess Le^Y expression, organoids were fixed in 10% formalin for at least 1 h, and then embedded in paraffin before performing immunohistochemistry for Le^Y, as described above.