Sn 38

After 3 days of culture, spheroids were washed in PBS, filtered, resuspended in fresh stem cell medium and counted. For each screen, approximately 1000 spheroids were added to an IndiTreat™ screening array (2cureX, Birkerød, Denmark) containing concentration gradients of 5-FU, oxaliplatin, SN38 (the active metabolite of Irinotecan) (Sigma-Aldrich, St. Louis, MO, USA) and combination treatments FOLFOX (5-FU + oxaliplatin) and FOLFIRI (5-FU and SN38). The arrays were scanned on screening day 0, 4 and 7 using an oCelloScope system (Phillips BioCell, Allerød, Denmark). The obtained images were analysed for changes in spheroid area using proprietary Phillips BioCell and 2cureX algorithms. For each well, the relative growth inhibition was calculated by dividing the total spheroid area with the area of the same well at day 0 and the average of the negative controls on the same day as the measurement day. Dose response curves, adjusted r² values and ED25 values were plotted and calculated using Matlab (MathWorks, Natik, MA, USA). Less than lowest dose or higher than highest dose was used in cases where ED25 values were calculated to be outside the used compound concentration ranges.

PDAC cell lines BxPc-3, MiaPaca-2, and Panc1 were purchased from ATCC and routinely screened for mycoplasma. The PDAC cell line L3.3 was a generous gift from John’s Hopkins University. All cells were cultured in DMEM (Corning) supplemented with 10% FBS (Atlas Biologicals), 1% penicillin-streptomycin, and 1% MEM nonessential amino acids (Corning). All PDAC cells tested were p53 mutants. Cells were maintained at 37˚C in an atmosphere containing 5% CO₂. AZD1775 was provided by AstraZeneca or purchased from MolPool (Hong Kong) depending on availability during the study. Irinotecan was purchased from the University of Colorado Hospital Pharmacy. The active metabolite of Irinotecan, SN38, was purchased from Sigma for in vitro analyses. Capecitabine and navitoclax were purchased from Active Biochem. The active metabolite of Capecitabine, 5-fluoruracil (5-FU), was purchased from the University of Colorado Hospital Pharmacy for in vitro analyses.

Anticancer drugs and positive reversal agents: Venetoclax was purchased from ChemieTek (Indianapolis, IN, USA). Paclitaxel, mitoxantrone, topotecan, SN-38, verapamil, KO143, and cisplatin were purchased from Sigma Co (St. Louis, MO, USA). Cell culture: Bovine serum (BS), fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM) and trypsin (0.25%) were purchased from Corning Inc. (Corning, NY, USA). Cell viability assay: dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Co. Immunoblotting: Human monoclonal antibodies for ABCG2 and GAPDH were purchased from Millipore (Billerica, MA, USA). Alexa Fluor 488 conjugated secondary antibody was purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA). Triton X-100, 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Co. Other chemicals: [³H]-labeled mitoxantrone (2.5 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). All other chemicals were purchased from Sigma Co.

A filter-sterilized stock solution of 10 mM 5-Fluorouracil (5-FU) (Sigma Aldrich, Diegem, Belgium) was prepared in dimethyl sulfoxide (DMSO). The stock solution was further diluted (1:1,000) in the mucin agar (see M-SHIME Experimental set-up) to a final concentration of 10 µM.
A filter-sterilized stock solution of 10 mM SN-38 (Sigma Aldrich, Diegem, Belgium) was prepared in DMSO. The stock solution was further diluted (1:1,000) in the medium to a final concentration of 10 µM.

Cell lines were obtained from the ATCC. HCT116 cells (ATCC Cat# CCL-247, RRID:CVCL_0291) were cultured in McCoy’s 5A medium supplemented with 10% FBS, 4 mM L-glutamine (Cat#03-020-1B, BI-Biologicals, Ingelheim, Germany), 2 mM L-alanyl-L-glutamine (Cat#03-022-1B, BI-Biologicals), and 1% penstrep (Cat#03-031-1B, BI-Biologicals) and were grown in a humidified incubator at 37 °C and 5% CO₂ [71 (link)]. SN-38 was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were seeded in quadruplicate in 96-well plates at a density of 2 × 10³ cells per well. At the indicated time, 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Saint-Quentin, France) were added and incubated at 37 °C for 4 h. Formazan crystals were solubilized in DMSO and absorbance read at 560 nm on a spectrophotometer. Results were normalized to the cell density at day 1. For cytotoxicity assays, cells were seeded in quadruplicate in a 96-well plate (2.5 × 10³ cells per well) and exposed the day after to increasing concentrations of cytotoxic drugs including 5-fluorouracil, SN38, oxaliplatin (Sigma-Aldrich, Saint-Quentin, France) or to vehicle alone. The cells were exposed to the drug during six days, and cell viability was quantified each day using MTT assay. Values were normalized to the mean optical density of the control for each day.