Rspo1

AM epithelial cells were seeded into 96-well culture plates in a density of 1 × 10⁴ cells/well in 100 µl of defined KGM-2 medium with five independent replicates per treatment condition. After 24 h, the cells were washed once with PBS and starved in Keratinocyte basal Medium 2 (KBM2, Lonza) overnight. Then Rspo1 (PeproTech) and Rspo2 (PeproTech) were administrated to the starved AM epithelial cells at concentrations of 0, 5, 10, and 20 ng/ml, respectively. After 72 h, 10 µl of CCK-8 reagent (Cell Counting Kit-8 assay, BioLegend) was added into each well and incubated at 37 °C for 2 h. For drug resistance testing, AM epithelial cells were seeded into 96-well plates (5 × 10⁴ cells/well) and treated with PLX4032 (Cayman Chemical Company) for 48 h and the cell viability was determined by Cell Count Kit-8. The absorbance at 450 nm wavelength was detected using an OPSYS Mr microplate reader (Thermo Fisher).

Surgical-resected PDAC specimens for organoid preparation were immersed in cold PBS saline (Basal Medium # B310KJ) with 1% Penicillin/Streptomycin (Basal medium # S110JV). After cutting and digestion via the Solo Digestive Enzyme Kit (Sinotech Genomics # JZ-SC-58201) for 1–2 h, the cell mixture was filtered through a 70 μm sieve and centrifuged at 300 g for 5 min. The pellet was embedded in GFR Matrigel (R&D systems #3533–005-02) and cultured in a complete organoid expansion medium containing RSPO1 (Peprotech #120–38), WNT3A (Peprotech #315–20), NOGGIN (Peprotech #120-10C), FGF10 (Peprotech #100–26), EGF (Peprotech # AF-100–15) and other supplements as previously described. Organoids were generated and passaged for 3–5 generations and then cryopreserved. The recovered organoids used in our study are between the 7th and 10th generations, and the culture medium is changed every 4 days. For co-culture of organoids and T cells, tumor organoids were partially digested from Matrigel to retain the three-dimensional architecture. The remaining Matrigel was washed before coculture. Cell count and viability were performed via ViaStainTM AO/PI staining (Nexcelom #CS2-0106) on living organoids. The stained organoids were imaged under Confocal laser scanning microscopy (Olympus FV3000) and quantified via Fiji (Image J) software.

Primary hepatocytes were cultured as 3D organoids according to the protocol set up by Huch and Clevers^{18 (link)}. Briefly, after centrifugation primary hepatocytes were mixed with growth factor reduced Matrigel (Corning), and a 50 μL drop of this solution was plated in the middle of each well of a 24-well plate. For the first set of experiments, comparing organoïds formation in three different batches of human hepatocytes (Fig. 1), 30,000 cells per well have been plated. For the other experiments, focusing on HEP187269, 300,000 cells per well were used. After solidification of Matrigel the EM expansion media was added, composed of AdDMEM/F12 (Life Technologies) supplemented with 1% N2 and 1% B27 (Life technologies), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), 10 mM Nicotinamide (Sigma), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), and the growth factors: 50 ng/mL EGF (R&D Systems), 500 ng/mL Rspo1 (Peprotech), 100 ng/mL FGF10 (Miltenyi Biotec), 25 ng/mL HGF (Miltenyi Biotec). During the 3 first days after plating, the media was supplemented with 25 ng/mL Noggin (Peprotech), 50 ng/mL Wnt3a (R&D Systems), and 10 μM Y27632 (Stem Cell Technologies).