Capan 1

Four human pancreatic cancer cell lines (BxPC-3, PANC-1, CAPAN-1 and AsPC-1) were purchased from American Type Culture Collection (Manassas, VI, USA) via In Vitro Technologies Pty Ltd. (Noble Park North, Australia). BxPC-3 and AsPC-1 cells were cultured using Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich Fine Chemicals, St Louis, MO, USA) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). CAPAN-1 cells were cultured using Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich Fine Chemicals, St Louis, MO, USA) supplemented with 20% FBS and 1% penicillin/streptomycin. PANC-1 cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, Castle Hill, NSW, Australia) supplemented with 10% FBS and 1% P/S. All cell lines were grown in T75 flasks and incubated at 37 °C in a 5% carbon dioxide in air atmosphere. Cells were routinely assessed for mycoplasma contamination and used within three months of receipt or resuscitation. For experiments, cells were grown to confluence then washed twice with phosphate-buffered saline (PBS) and detached from the flask by TrypLE Select Enzyme (1X) (Thermo Fisher Scientific Australia Pty Ltd., Scoresby, Australia). All cell culture reagents and culture media were purchased from Sigma-Aldrich Pty Ltd. (Castle Hill, Australia) unless otherwise stated.

The human colorectal carcinoma cell line HCT116 was kindly provided by Dr. Vogelstein from Johns Hopkins University, Baltimore. The pancreatic adenocarcinoma cell line Capan-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCT116 cells were cultured in McCoy’s 5A Modified Media (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, PAA, Linz, Austria) and 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA). Capan-1 cells were grown in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS. BOLD-100-resistant HCT116 and Capan-1 sublines were generated over several months by regular exposure to increasing concentrations of BOLD-100, followed by a drug-free recovery phase. Finally, BOLD-100-resistant HCT116 (HCTR) and Capan-1 (CapanR) cells were selected with 200 µM BOLD-100 in two-week-intervals. Resistance was maintained for >1 month without selection pressure. All cell cultures were grown in 5% CO₂ at 37 °C and regularly checked for Mycoplasma contamination.

Human pancreatic carcinoma cell lines Panc-1 (HER2-low-expressing cell line), Capan-1 (HER2-high-expressing cell line), and the NK cell-sensitive thymoma cell line K562 were purchased from the American Type Culture Collection (Manassas, VA). HER-2 expression on the surface of Panc-1 and Capan-1 was confirmed by flow cytometry (Fig 1A). All these cell lines were maintained according to the manufacturer’s instructions. In brief, Panc-1 was cultured with Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life Technologies, Santa Clara, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 10% penicillin and streptomycin solutions (Gibco). Capan-1 was cultured with ISCOVE modified Eagle’s medium (Sigma Chemical Company, St. Louis, MO) supplemented with 10% FCS. Cells were removed by short-term incubation with trypsin-EDTA (Gibco) and about one-third to one-fourth of the viable cells were removed and resuspended in fresh culture medium twice weekly. K562 was cultured in DMEM supplemented with 10% heat-inactivated FCS, HEPES-buffer solution 5 mM, penicillin and streptomycin solutions 100 U/ml, L-glutamine 2 mM, sodium pyruvate solution 2 mM, and nonessential amino acid solution 2 mM (all purchased from Gibco-BRL), modified vitamins 2 mM (DS Pharma, Osaka, Japan), and 2-mercaptoethanol 2 mM (Sigma Chemical).