Organoid Culture and Viability Assay

Organoids were cultured as previously described (24). Briefly, surgically removed cervical tumors were cut into small pieces of tumor tissue. Fresh ascites from ovarian cancer patients was centrifuged to obtain cell pellets. Tumor tissues or cell pellets were then digested with Collagenase Type II (Gibco, USA) followed by the treatment with RBC Lysis Buffer (Bio-Legend, USA). After centrifugation, tissues/cell pellets were suspended in AdDF+++ [Advanced DMEM/F12 containing 1% HEPES (Gibco, USA), 1 x GlutaMAX (Gibco, USA) and antibiotics] and pipetted repeatedly with a syringe, filtered through a 100 μm filter. The isolated cells were mixed with Growth Factor-reduced Matrigel (BD Biosciences, USA) and dropped on 24-well plates. On Matrigel stabilization, the organoid culture medium [AdDF+++ containing 10 ng/ml Noggin (PeproTech, USA), 10 ng/ml Rspo1 (PeproTech, USA), 1.25 mM N-Acetylcysteine (Sigma, USA), 10 mM Nicotinamide (Sigma, USA), 0.5 μM TGFβ Receptor inhibitor A83-01 (Sigma, USA), 10 ng/ml FGF10 (PeproTech, USA), 37.5 ng/ml Heregulin β-1 (PeproTech, USA), 5 μM RhoK inhibitor Y-27632 (AbMole Bioscience, USA), 5 ng/ml EGF (PeproTech, USA), 10 μM Forskolin (Bio-Techne, USA), 500 ng/ml Hydrocortisone (Sigma, USA), 100 nM β-Estradiol (Sigma, USA), 2% B27 supplement (Gibco, USA), and 0.2% Primocin (In vivoGen, USA)] was added and the plates were transferred to humidified 37°C/5% CO₂ incubators. For passaging, organoids were digested with TrypLE Express Enzyme (Thermo Fisher Scientific, USA) and re-inoculated to form new organoids. The organoid culture medium was changed every 3 days and passaged every 1 to 4 weeks. Organoid structures were imaged by an inverted phase-contrast microscope (Leica Microsystems, Germany).

For organoid viability assay, organoids were dissociated and cultured in 96-well plates followed exposure to the organoid culture medium containing PEITC and/or BMN 673. The organoid culture medium was changed every 3 days. The ATP levels were measured with CellTiter-Glo^® 3D Reagent (Promega, USA) according to the manufacturer’s instructions. Luminescence signals were measured using a SpectraMax microplate reader (Molecular Devices, Austria).