R spondin1

CoCaB 1 PDX chunks were minced to 1 mm pieces with a sterile scalpel in PBS and incubated in 5 mg/ml Type II Collagenase (Gibco, Waltham, MA) in Advanced Dulbecco’s modified Eagle’s medium (DMEM)/F12 for 1 h at 37 °C, followed by 5 min further digestion with 0.25% trypsin. The tissue-cell mixture was passed through a 40um nylon mesh to obtain single cells. Twenty thousand cells were embedded in 50 µl Matrigel (Corning) and cultured in complete medium containing B-27 supplement, 1.25 mM N-Acetyl-L- cysteine (Sigma-Aldrich, St. Louis, MO), 50 ng/ml EGF (Peprotech, Rocky Hill, NJ), 200 nM A83-01 (Tocris Bioscience, UK), 500 ng/ml R-spondin1 (conditioned medium and R-spondin1 expressing plasmid provided by Yu Chen, Memorial Sloan Kettering Cancer Center), 10 µM Y- 27,632 (Sigma-Aldrich), and 100 ng/ml Noggin (Peprotech) in 24-well ultra-low attachment plates (Corning) as previously described^{20 (link)}.

Intestinal duodenal and colonic organoids were generated and cultured as described (Degirmenci et al., 2018a (link), 2018b (link); Sato and Clevers, 2013 (link); Valenta et al., 2016 (link)). Organoids were cultivated in medium supplemented as follows: duodenal organoids in ENR = EGF (Gibco/Thermofisher) 50 ng/mL, Noggin (Sigma) 100 ng/mL, R-spondin1(Sigma) 500 ng/mL; colonic organoids in ENRW = ENR + 30% Wnt3a conditioned medium, tumor organoids N= Noggin (Sigma), 100 ng/mL. Organoids from AOM/DSS tumors were generated from 3 months old AOM/DSS tumors as described by Xue and Shah (2013) and cultured for first week full medium for colon organoids (ENRW), followed by cultivation in medium lacking Wnt3a, R-spondin1 and EGF. The tumor organoids can grow in such a medium for minimally 4 weeks. Tumor organoids were generated from tumors of Wls^flox/flox and Villin-Cre^ERT2,Wls^flox/flox animals. To achieve Cre-mediated recombination and thus Wls elimination control (Wls^flox/flox) and Villin-Wls^cKO (Villin-Cre^ERT2,Wls^flox/flox) organoids were treated with (Z)-4-Hydroxitamoxifen (4-OHT, Sigma), 500 ng/mL 24 hour after passaging for 12 hours then let to grow till splitting, passaged and treated again with 4-OHT for 12 hours.

For identifying the HM-SE genes, 2 patients with PT and 2 patients with HM were enrolled for H3K27ac ChIP-Seq analysis (Table S1). The samples were obtained from Ruijin Hospital, Shanghai Jiaotong University School of Medicine. The study protocol was approved by the Research Ethics Committee of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University. All enrolled participants consented to attend this cohort study and signed written informed consent. To create the metastatic patient-derived organoids (MDO), the fresh ccPDAC tissues were enzymatically digested and then treated for 1 hour at 37°C with 200 U/ml of deoxyribonuclease I (Roche) and collagenase type IV (SigmaAldrich) (Table S1). The cells plated in Basement Membrane (OuMel, #WM-MG-01) were filtered using a 70-m nylon mesh and and cultured by DMEM/F12 media containing 2% B27, N-acetyl-l-cysteine (SigmaAldrich, 1.25 mM), EGF (Gibco, 50 ng/mL), A83-01 (SigmaAldrich, 200 nM), Noggin (SigmaAldrich, 100 ng/mL), R-spondin 1 (SigmaAldrich, 500 ng/mL), Y-27632 (MedChemExpress, 10 mM), and dihydrotestosterone (SigmaAldrich, 1 nM).