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Murine pancreatic primary tumor organoids (mT3, mT6, mT19, and mT23), metastatic organoids (mM1, mM3, mM6, and mM10), and tumor 2D cell lines (mT3‐2D, mT4‐2D, mT5‐2D, and mT8‐2D) from the tumor‐bearing KPC mice were established and characterized previously.^[6 (link),
⁸ (link),
⁶⁷ (link)
^] Murine pancreatic organoid culture media contains Advanced DMEM/F‐12 (Thermo Fisher 12 634 028), 10 mm HEPES (Thermo Fisher 15 630 080), 1% Penicillin‐Streptomycin (Thermo Fisher 15 140 122), 1% GlutaMAX Supplement (Thermo Fisher 35 050 061), 0.5εm A 83‐01 (Fisher Scientific 29‐391‐0), 0.05 µg mL⁻¹ mEGF (Fisher Scientific PMG8043), 0.1 µg mL⁻¹ hFGF‐10 (Pepro Tech 100–26), 0.01εm hGastrin I (Fisher Scientific 30–061), 0.1 µg mL⁻¹ mNoggin (Pepro Tech 250‐38), 1.25 mmN‐Acetyl‐L‐cysteine (Millipore Sigma A9165), 10 mm Nicotinamide (Millipore Sigma N0636), 1X B‐27 Supplement (Fisher Scientific 17‐504‐044), and 1x RSPO1‐conditioned medium. Murine 2D culture media contains DMEM (Corning 10‐013‐CV), 10% FBS (Gen Clone 25–550H), and 1% Penicillin‐Streptomycin. Human PDA cell lines SUIT2 (Glow Biologics GBTC‐1088B), CFPAC1 (ATCC CRL‐1918), BxPC3 (ATCC CRL‐1687), and PATU 8988s (Glow Biologics GBTC‐0209H) were cultured with RPMI 1640 (Corning 10‐040‐CV), 10% FBS, and 1% Penicillin‐Streptomycin.

Organoids were grown media composed of Advanced DMEM/F12 (Thermo Fisher) supplemented with 100 µ/mL Primocin (InvivoGen), 1x GlutaMAX (Thermo Fisher), 1x B27 supplement (Thermo Fisher), 10 mM Nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 nM Gastrin (Sigma-Aldrich), 100 ng/mL hFGF-10 (Peprotech), 50 ng/mL EGF (Peprotech), 100 ng/mL mNoggin (Peprotech), 500 nM A83-01 (Tocris biosciences), 10% (v/v) R-Spondin-1 conditioned medium, and 50% (v/v) Afamin-Wnt3a conditioned medium⁴³ . All conditioned mediums were generated in-house by the Cell Services Laboratory. Human organoids were passaged using TrypLE Express (Gibco) and resuspended in Matrigel (Corning). Organoid medium was supplemented with 10 µM Y-27632, which was removed upon the first medium change (2–3 days).

PDOs were obtained from the PDAC organoid biobank of the Department of Visceral, Thoracic and Vascular Surgery at the University Hospital Carl Gustav Carus of the TU Dresden (VTG) [27 (link),40 (link)]. Initial establishment of PDOs from resection material or fine needle aspiration was described in an earlier study [40 (link)]. The PDO generation and cultivation were approved by the local ethics committee at Technische Universität Dresden, Germany (#EK451122014). PDOs were embedded in GFR Matrigel (Corning, Corning, NY, USA) and cultured in PDAC culture media based on DMEM/F12 (Gibco, Waltham, MA, USA) supplemented with Pen/Strep (1%, Gibco), GlutaMAX (1%, Gibco), HEPES (1%, Gibco), Wnt3a (100 ng/mL, R&D Systems), R-Spondin (250 ng/mL, PeproTech, Hamburg, Germany), Noggin (100 ng/mL, PeproTech), B27 (1×, Gibco), Nicotinamid (10 mM, Sigma-Aldrich, St. Louis, MO, USA), Gastrin (10 nM, Sigma-Aldrich), N-actyl-L-cysteine (1.25 mM, Sigma-Aldrich), Primocin (100 µg/mL, InvivoGen, San Diego, CA, USA), mEGF (50 ng/mL, Sigma-Aldrich), A-83-01 (0.5 µM, Tocris, Bristol, UK), recombinant human fibroblast growth factor 10 (hFGF-10, 100 ng/mL, PeproTech), murine recombinant epidermal growth factor (mEGF, 50 ng/mL, Sigma-Aldrich) and N2 (1×, Gibco). PDOs were passaged once (DD1522) or twice (DD442, DD314, DD1521) a week as described previously [25 (link)] with a split ratio of 1:2.