Human platelet growth factor

Human metastatic pancreatic cancer L3.6pl cells were plated in six-well ultra-low attachment plates (Corning Inc., Corning, NY) at a density of 1,000 cells/mL in DMEM supplemented with 1% N2 Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/mL human platelet growth factor (Sigma-Aldrich), 100 ng/mL epidermal growth factor (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Spheroids were collected after 7 days and dissociated with Accutase (Innovative Cell Technologies). Fluorescence-activated cell sorting experiments were carried out by flow cytometry using antibody against stem cell markers CD44-APC, CD24-FITC, and CD133-PE.

For sphere formation assays, cells were seeded in 6-well ultralow attachment plates at a density of 1 × 10³ cells/mL. Add Dulbecco’s modified Eagle’s medium containing 2% B27 supplement (Invitrogen, Carlsbad, CA, USA), 1% N2 supplement (Invitrogen, Carlsbad, CA, USA), 100 ng/mL epidermal growth factor (Invitrogen, Carlsbad, CA, USA), 20 ng/mL human platelet growth factor (Sigma-Aldrich, St. Louis, Missouri, USA), and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA). Then incubate in 37 °C, 5% CO₂ and humidified atmosphere of 95% air. After 7 days, single spheres were chosen and counted.

Spheroid forming assays were performed as described. In brief, cells were plated in six-well ultralow attachment plates (Corning Inc., Corning, NY) at a density of 1,000 cells/ml in DMEM supplemented with 1% N2 Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Spheroid were collected after 7 days and dissociated with Accutase (Innovative Cell Technologies, Inc.). The cells obtained from dissociation were sieved through a 40-μm filter, and counted by coulter counter using trypan blue dye.

Pancreatic cancer cell lines AsPC‐1 and PANC‐1 were used, and these cells were purchased from American Type Culture Collection (Manassas, Virginia) purchase. Cells were grown and frozen in liquid nitrogen for future use. Cells from second and third passages were used for the experiments. ATCC utilizes short tandem repeat (STR) profiling to authenticate the cell lines. PANC‐1 possesses mutations in p53 and K‐ras genes in codon 273 and codon 12 respectively. AsPC‐1 harbours mutation on codon 12 of K‐ras gene. CD133+/CD44+/CD24+/ESA+human pancreatic CSCs were isolated from primary tumours as described previously.26 Pancreatic CSCs isolation and characterization from KrasG12D mice were performed as described elsewhere.26 Pancreatic CSCs were grown in specialized growth medium (Celprogen, Inc, Torrance, California) which contained 1% N2 Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/mL human platelet growth factor (Sigma‐Aldrich), 100 ng/mL epidermal growth factor (Invitrogen) and 1% antibiotic‐antimycotic (Invitrogen). Pancreatic CSCs and cancer cell lines were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂.