Cell viability assays

Cells were plated in 96-well plates in the presence of treatment and medium containing SYTOX Green (125 ng/ml) and evaluated in the IncuCyte (Essen BioScience) for 72 hours in 1 or 21% O₂. Following the 72-hour incubation period, HCS NuclearMask red dye was added to the wells to obtain an accurate number of cells remaining in each well. To assess cell viability, cytotoxic indeces were calculated as follows: (number of SYTOX Green–positive cells per mm² ÷ number of HCS NuclearMask red cells per mm²) × 100. The following processing definition parameters were used in the analysis of the IncuCyte data: SUM159PT/UC-16/4T1: green, Top-Hat, radius of 100 μm, threshold [green calibrated unit (GCU)] of 8, and filters with an area of 10 to 1000 μm²; red, Top-Hat, radius of 100 μm, threshold (GCU) of 50, and filters with an area of 50 μm² (minimum); LM2-4: green, Top-Hat, radius of 100 μm, threshold (GCU) of 8, and filters with an area of 10 to 1000 μm²; red, Top-Hat, radius of 100 μm, threshold (GCU) of 30, and filters with an area of 50 μm² (minimum); PK-8: green, Top-Hat, radius of 100 μm; threshold (GCU) of 8, and filters with an area of 100 to 1600 μm²; red, Top-Hat, radius of 100 μm, threshold (GCU) of 12, and filters with an area of 100 μm² (minimum); HT-29: green, Top-Hat, radius of 100 μm, threshold (GCU) of 10, filters with an area of 10 to 1000 μm², and eccentricity of 0.8 (maximum); red, Top-Hat, radius of 100 μm, threshold (GCU) of 50, filters with an area of 50 μm² (minimum), and eccentricity of 0.92 (maximum).

For drug screening, Greiner 96-well plates were coated by diluting Matrigel 1:30 with organoid basal medium (66) and plating 100 μl per well. The Matrigel was allowed to polymerize for at least 1 hour before the remaining medium was removed and complete organoid medium containing 5000 cells per well was plated over. The organoids were allowed to grow for 96 hours before drugs were added in an additional 100 μl of organoid medium per well in triplicate for 72 hours. Organoids were stained with ethidium homodimer and Hoechst for 1 hour and scanned on an IN Cell Analyzer 2200 instrument. The resulting images were counted using IN Cell Analyzer 2200 software, and data were analyzed using the IN Cell Analyzer Workstation 3.7.3 software.