Pmg8043

Organoids were derived from pancreatic tumors of KPC (Kras^LSL-G12D/+;Trp53^LSL-R172H/+;Pdx1-Cre) mice in a C57BL/6 background and described before (23 ). Organoids were cultured 24-well plates in growth factor reduced (GFR) Matrigel (Corning) in Advanced DMEM/F12 supplemented with the following: 1% P/S, 2 mM glutamine, 1X B27 supplement (12634–028, Invitrogen), 50 ng/ml murine EGF (PMG8043, Peprotech), 100 ng/ml murine Noggin (250–38; Peprotech), 100 ng/ml human FGF10 (100–26; Peprotech), 10 nM human Leu-Gastrin I (G9145, Sigma), 1.25 mM N-acetylcysteine (A9165; Sigma), 10 mM nicotinamide (N0636; Sigma), and R-spondin1 conditioned media (10% final). Organoids were passaged with every 3–4 days. For PSC co-culture, confluent wells of organoids were dissociated with 1x TrypLE (12604013, Thermo Fisher) and plated at a splitting ratio of 1:5 (approximately 1×10⁴ cells) together with 8×10⁴αSMA-DsRed expressing PSCs in GFR Matrigel. Co-cultures were cultured with DMEM supplemented with 10% FBS (Gemini) and 1% P/S in 20% O₂ and 5% CO₂. For experiments in hypoxia, co-cultures were placed in a hypoxia glove box (Coy) set at 0.5% O₂ for the last 48h of the experiment. For measurement of organoid growth in co-cultures, organoids expressing Luciferase were used. Cultures were treated with Luciferin, lysed and luminescence was measured.