Rhfgf 10

All experiments with human material were performed in accordance with the Declaration of Helsinki and were approved by the ethics committee of the Medical Faculty of Heidelberg University (323/2004, Amendment 03). Informed consent was received from participants before study inclusion. Generation and cultivation of patient-derived PDAC cultures have been described previously (16 (link)). Cultures were subjected to SNP typing and Multiplex Cell Contamination Testing (Multiplexion). Cultures were grown in DMEM Advanced F12 medium supplemented with 0.6% (w/v) glucose, 2 mM L-glutamine, 2% B27 supplement (1×) (all Thermo Fisher Scientific), 12 μg/ml heparin and 5 mM HEPES buffer (both Sigma Aldrich), 10 ng/ml rhFGF basic, 20 ng/ml rhFGF-10, and 20 ng/ml rhNodal (all R&D Systems). Cytokines were renewed twice per week.

For sphere formation, the growth factor-reduced Matrigel was mixed with cells suspended in an equal volume of Advanced DMEM/F-12 media. The mixtures (60 μL) were placed around the bottom rim of each well in 48-well plates. After solidification at 37 °C for 20 min, each well was overlaid with 300 μL of Advanced DMEM/F-12 media supplemented with recombinant human (rh) EGF (50 ng/mL, Sigma, St. Louis, MO, USA), rhR-spondin I (500 ng/mL, R&D systems, Minneapolis, MN, USA), rhFGF10 (50 ng/mL, R&D systems), recombinant mouse Noggin (100 ng/mL, R&D systems), and 10 mM Nicotinamide. Media was changed twice per week. The sphere numbers were counted, and fluorescence images were taken using a Lecia DFC365 FX microscope. Images were analyzed with Leica Application Suite X v1.9.0 and Image J v1.46r.

CRPC with docetaxel chemotherapy-resistant patient-derived organoids was established pursuant to the method as described previously [51 (link),52 (link)]. The organoids were cultured with the advanced DMEM/F12 supplemented with 125 ng/ml rhR-spondin-1 (STEMCELL Technologies, 78213.1), 100 ng/ml rhNoggin (R&D Systems, 6057-NG-025), 1 ng/ml rhFGF (R&D Systems, 233-FB-025), rhFGF-10 (R&D Systems, 345-FG-025), 50 ng/ml (Meilunbio, China, MB8218-1), 1×N21 (R&D Systems, AR008), 10 μM Y-27632 (Abcam, Ab120129), 0.5 μM A83-01 (Beyotime Biotechnology, SF7917), 1:100 primocin (Invivogen, ant-pm-1), 10 μM SB202190 (Beyotime Biotechnology, SC0380), 10 mM Nicotinamide (Beyotime Biotechnology, S1761), and 1.25 mM N-acetylcysteine (Selleck, S1623), and the medium was changed every 2 to 3 days. Lentiviral transduction of AZGP1P2 shRNA and shRNA control was conducted according to the method as previously described [53 (link)]. The proliferation of organoids treated with 20 nM docetaxel was assessed by CCK-8 assay on the fifth day, and organoids were photographed using light microscopy. Three independent experiments were performed.