Cell Viability and Analysis for Dose Titration Experiments

Cell viability was analyzed 6 days after initial treatment (Day 7) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (LifeTechnologies, Catalog Number M6494) similar to our prior studies^21,29. Briefly, microtumors were transferred to 96-well plates using an electronic dispenser with wide-mouth pipet tips (ViaFlo) to ensure that each well in the 96-well plate contained one microtumor bead, and each condition had at least three replicates. MTT was prepared, and the manufacturer’s protocol was followed for lysis, dissolution, and analysis, measuring absorbance at 570 nm. Raw MTT absorbance data was obtained, and average background control was subtracted from raw data using a Synergy HT fluorescence plate reader (Biotek). Dose response data was analyzed using GraphPad Prism version 4 (GraphPad Software, San Diego, CA), and dose response curves were assembled by normalizing the absorbance to DMSO control and plotted as bar graphs. From the dose finding experiments and acquired dose response curves, the IC₅₀ means and standard deviations were calculated via GraphPad Prism using non-linear regression curve fitting approach. Dose titration curves and raw data were used to determine the average IC₅₀s for each drug, excluding selumetinib for which an “IC₅₀*” dose was selected (due to low efficacy, true IC₅₀ could not be determined), for subsequent single and combination drug screening.