Cell viability testing

Cell viability in spheroids was assessed using the Cell-Titer-Glo™3D reagent (Promega) following the manufacturer’s protocol. Briefly, 100 μl of culture medium was replaced with an equal volume of room temperature Cell-Titer-Glo™3D reagent. After mixing followed by 30 min incubation, under agitation in the dark, the contents of each well were transferred to a white, flat bottom 96-well plate and the luminescent signal in each well was measured using a spectrophotometer multi-plate reader (FluoStar, Omega, BMG Labtech, Ortenberg, Germany). Cell viability was assessed at the end of volume growth monitoring (at 21 days) or 4 days after treatment for a subset of thermal/radiation doses. Viability is reported relative to untreated controls, and all data are the means and standard deviations of at least three experiments (n≥3). Statistical analysis was performed in GraphPad Prism using an unpaired t-test.