Cell Viability Evaluation in 2D and 3D

2D cell viability following 48–72 h drug treatment was evaluated using CellTiter-Glo reagent (Promega) according to manufacturer’s instructions. Assays were performed in triplicate and experiments were repeated three times in all five cell lines. Spheroids were successfully developed in three of the cell lines, UOK109, UOK120, and UOK124, using a previously described methodology [24 (link)] to evaluate 3D cell viability. Spheroids were incubated with drug for five days, before assessing viability by CellTiter-Glo 3D Cell Viability Assay (Promega). Calculation of drug synergy was performed with Compusyn [25 (link)], according to software instructions, including at least 6 concentration points for each drug alone and in combination. Cell cytotoxicity in vitro was measured with the lactate dehydrogenase (LDH)-based Cytotoxicity Detection Kit (Roche) as previously described [26 (link)].