Panc 1

Human pancreatic cancer cell panc-1 was obtained from American Type Culture Collection (ATCC Manassas, VA, USA) and cultured in Dulbecco Modified Eagle Medium (DMEM, Gibco, Paisley, UK), supplemented with 10% fetal bovine serum (FBS, Gibco, Paisley, UK), 100 U/ml pencillin and 100 U/ml streptomycin at 37°C incubator with 5% CO2. For isolating CSCs sub-population from panc-1 cells, panc-1 cells were maintained in DMEM/F12 without FBS, supplemented with 2% B27 supplement (Life Technologies, Grand Island, NY, USA), 20 ng/ml human EGF (PeproTech, Rocky Hill, NJ, USA.), 40 ng/ml bFGF (PeproTech, Rocky Hill, NJ, USA.) and 5ug/ml insulin (PeproTech, Rocky Hill, NJ, USA.). Under this culture condition, non-adherent cell aggregates referred to as spheres were formed. The medium was half-refreshed every 2–3 days. 10–14 days is required to form obvious spheres.

MIA PaCa-2, PANC-1, HT-29, SW480, LN-18 and LN-229 cells were obtained from America Type Culture Collection, (ATCC, Manassas, VA; #CRL-1420, CRL-1469, HTB-38, CCL-228, CRL-2610 and CRL-2611, respectively). MIA PaCa-2, PANC-1, HT-29 and SW480 were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA), supplemented with 2mM L-Glutamine (Life Technologies), 10% HyClone fetal bovine serum (FBS, GE Healthcare, Piscataway, NJ) and 100 U/mL penicillin and 100 μg/mL streptomycin (referred to as 1% Pen/Strep, Life Technologies). PANC-1, LN-18 and LN-229 were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 1% Pen/Strep. All cell lines were maintained at 37°C, 5% CO₂, 85% RH,routinely tested for mycoplasma contamination and authenticated by short tandem repeat (STR) profiling.

The human lung adenocarcinoma cell line, HCC827, the human pancreatic ductal adenocarcinoma cell line, PANC-1, and human the colorectal adenocarcinoma cell line, SW620, were purchased from ATCC (Manassas, VA) and maintained mycoplasma free at passage numbers <25 for all studies. The cell lines were expanded in their respective optimal growth media (HCC827: RPMI 1640 [ThermoFisher Scientific] + 10% fetal bovine serum [FBS] + 1% penicillin/streptomycin/glutamine; PANC-1: DMEM [ThermoFisher Scientific] + 10% FBS + 1% penicillin/streptomycin/glutamine; SW620: Leibovitz L-15 [ThermoFisher Scientific] + 10% FBS + 1% penicillin/streptomycin/glutamine) and stored at 37 °C in either a 5% (HCC827 and PANC-1) or 0% (SW620) CO₂ incubator.

Porcine kidney tubular epithelium nucleoside transporter deficient cells (PK15NTD) were kindly donated by Dr. Chung-Ming Tse (Johns Hopkins University, Baltimore, MD). Human pancreatic cancer Panc-1, HPAC II and MIA PaCa-2 cell lines were purchased from ATCC (Manassas, VA). Cells were maintained in Eagle’s minimal essential medium/Earle’s Balanced Salt Solution with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (PK15NTD), Dolbeco’s minimum essential medium (Panc-1 and MIA PaCa-2) and 10% fetal bovine serum (Invitrogen, Grand Island, NY) and 2.5% horse serum additionally for MIA PaCa-2), Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 0.002 mg/ml insulin, 0.005 mg/ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor and 5% fetal bovine serum (HPACII) at 37 °C in a humidified atmosphere of a mixture of 5% CO₂ and 95% air.