Organoid culture

Mouse pancreatic organoids (StemCell Technologies #70933) were cultured in PancreaCult Organoid Growth Medium (StemCell Technologies #06040) according to the manufacturer’s protocol.

Patient-derived pancreatic cancer organoids were established from fresh surgical specimens obtained from patients who underwent surgical resection at Kyoto University Hospital, approved by the Ethics Committees (R1281) and by the Ethical Committee of Sumitomo Pharma (2017–04). The pathological characteristics of the primary tumor are presented in Table 1. Primary tumor tissue samples were processed as previously reported, with some modifications [7, 8, 14]. Briefly, the cell aggregates were embedded in Matrigel (Corning, Cambridge, MA, USA) and covered by a medium composed of 50% L-WRN conditioned medium (ATCC) containing L-Wnt3A, R-spondin 3, and Noggin, consisting of Advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 5% FBS, 2 mmol/l L-Alanyl-L-Glutamine (Wako, Tokyo, Japan), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Nacalai Tesque), 2.5 μg/ml Plasmocin prophylactic (Invitrogen), 10 μM Y-27632 (Tocris Bioscience), 1x B27 Supplement (Thermo Fisher Scientific, Waltham, MA, USA), 1 μM SB431542 (Tocris Bioscience), 100 ng/ml recombinant human fibroblast growth factor-basic (bFGF; Thermo Fisher Scientific), and 20 ng/ml recombinant human epidermal growth factor (EGF; Thermo Fisher Scientific). After confirming several passages of the PDOs, the organoids were also cultured with the following “complete medium” consisting of Advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 2 mM Glutamax-I (Wako, Tokyo, Japan), 10 mM HEPES (Thermo Fisher Scientific), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Nacalai Tesque), 10 μM Y-27632 (Tocris Bioscience), 1x B27 Supplement (Thermo Fisher Scientific, Waltham, MA, USA), 1 μM inhibitor of transforming growth factor-β (TGF-β) type I receptor, SB431542 (Tocris Bioscience), 50 ng/ml Wnt3A(R&D systems), 500 ng/ml R-spondin-1 (Peprotech Inc), 100 ng/ml Noggin (R&D systems), 100 ng/ml bFGF (Peprotech Inc), and 50 ng/ml EGF (Peprotech Inc). For culture of SMAD4-mutants, Sph18–06 was cultured in the complete medium without SB431542 (Tocris Bioscience). The passage number of PDOs was as follows: for in vitro experiments, Sph18–02 (≥P19), Sph18–06 (≥P8), Sph18–14 (≥P23), Sph18–21 (≥P12), Sph18–25 (≥P12), Sph19–07 (≥P12), Sph19–14 (≥P10), Sph19–22 (≥P6); and for in vivo transplantation experiments, Sph18–02 (≥P25), Sph18–06 (≥P16), Sph18–14 (≥P31), Sph18–21 (≥P31), Sph18–25 (≥P28), Sph19–07 (≥P19), Sph19–14 (≥P15), Sph19–22 (≥P16). Cell proliferation of PDOs was examined by seeding the same number of cells in triplicate and counting the cell number at day 7 using a Countess II FL automated cell counter (Thermo Fisher Scientific). Bright field images of PDOs were taken on an inverted microscope system (Olympus, IX73, 10x or 20x objective lenses).

Additional data that provide clinical information about the established PDOs

Values in CA19–9 indicate U/mL. Values in DFS and OS indicate months

Abbreviations: M male, F female, OS overall survival, DFS disease-free survival, mod moderately differentiated adenocarcinoma, poor poorly differentiated adenocarcinoma, AJCC American joint committee on Cancer, UICC International Union against Cancer, CA19–9 carbohydrate antigen 19–9, GEM gemcitabine, IMRT intensity-modulated radiotherapy, S-1 Tegafur, Gimeracil, Oteracil potassium, IPMN Intraductal papillary mucinous neoplasm, GnP gemcitabine and nab-paclitaxel, NA data not available, chemo chemotherapy, iv intravenous injection, CPT11 irinotecan

*M1 by peritoneal dissemination, **M1 by metastasis to para-aortic lymph node

For evaluation of effects of kinase inhibitor compounds on PDOs, cells of PDOs, Sph18–06 and Sph18–14, were dissociated, and the same number of cells (1 × 10³ cells/well) were plated in each of 384-well plates. After three days in culture, compounds from kinase inhibitor libraries (Selleck chemicals, L1200 and L2000) were added and further cultured for five days. Cell viability was examined by CellTiter-Glo 3D Reagent (Promega) according to the manufacturer’s instructions.