Gemcitabine hydrochloride

For in vitro assay, organoids were collected and dissociated into single cells by digesting with Accumax. The dissociated cells were counted using a TC20 Automated Cell Counter (Bio‐Rad, Hercules, CA, USA). For cell proliferation assays, 2 × 10⁴ single cells/well were plated in a 24‐well plate with solidified Matrigel to form organoids, or an ultra‐low attachment 24‐well plate to generate spheroids. Cell viability was analyzed with CellTiter‐Glo3D Cell Viability Assay (Promega, Fitchburg, WI, USA) at 5 time points (day 0, 3, 7, 10, and 14) in triplicate, and the results were normalized to the values on day 0. For the drug sensitivity assay, 5 × 10³ single cells/well were plated into PrimeSurface 96U (Sumitomo Bakelite, Tokyo, Japan) in triplicate. At 48 hours after plating, paclitaxel (Wako, Osaka, Japan), cisplatin (Wako), gemcitabine hydrochloride (Wako), crizotinib (Tocris Bioscience, Bristol, UK), and SU11274 (Cayman Chemical, Ann Arbor, MI, USA) were dispensed at 5 serially diluted doses from 5 to 100 nmol/L, from 1 to 100 μmol/L, from 1 to 100 nmol/L, from 100 nmol/L to 10 μmol/L and, from 100 nmol/L to 10 μmol/L, respectively, and analyzed in triplicate following 96 hours of drug incubation. The mean ± SD of the results from 3 independent experiments is shown for each drug.

The following items were purchased: fetal bovine serum (FBS), DMEM, RPMI, MEM and trypsin solution from Gibco (Life Technologies); RNeasy Mini kit from Qiagen (Hilden, Germany); High Capacity RNA-to-cDNA kit from Applied Biosystems (Darmstadt, Germany); NE-PER Nuclear and Cytoplasmic Extraction reagents and SuperSignal West Pico and Femto chemiluminescent substrates from Thermo Fisher Scientific (Rockford, IL, USA); FuGENE HD Transfection reagent from Promega (Madison, WI, USA); G418 (Geneticin) from Roche Diagnostics Deutschland GmbH (Mannheim, Germany); laminin, collagen from human placenta type IV [(collagen IV) from Sigma-Aldrich]; Cell Counting kit-8 from Dojindo Laboratories (Kumamoto, Japan); BioCoat Matrigel Invasion Chamber (8-µm pore size) and fibronectin from BD Biosciences (Franklin Lakes, NJ, USA); gemcitabine hydrochloride from Wako Pure Chemical Industries (Osaka, Japan); Fluorescent Mounting Medium from Dako Japan (Tokyo, Japan); Histofine Simple Stain Max PO (M) or (R) kit from Nichirei Biosciences, Inc., (Tokyo, Japan); and recombinant human epidermal growth factor (EGF, 236-EG) and recombinant human platelet-derived growth factor (PDGF, 220-BB) from R&D Systems (Minneapolis, MN, USA).