(A) Scheme of image-processing workflow. Multiple z-stacks were acquired in a tile-scan mode. H2B-mNeonGreen and bright field images were recorded of >10 organoids (left panel) over multiple days. Lower half of the imaged z-planes were selected of 3D-organoids that were fully recorded on each day (second panel). Live nuclei and dead nuclear remnants were marked for each z-plane, as identified by nuclear size (third panel, see Materials and methods section), measured and integrated per lower half of the 3D scanned organoid as an absolute measure for the amount of viable cells, while summed dead nuclei represent the amount of dead cells (fourth panel). (B) Bar diagrams showing proliferation and/or death of organoid cells following drug treatment and during recovery after drug removal. 3D tile-scans were acquired at the beginning and end of the therapy (day −3 and 0), as well as 3 and 7 days after the end of the therapy (i.e. drug removal) (day 3 and 7). All bars report pixel count from H2B-NeonGreen in living (color) as well as dead (black) organoid cells. Color corresponds to targeted inhibitor (see legend). All values are means ± s.e.m. of 12–15 organoids, normalized to ‘alive H2B’ prior to treatment. One representative z-plane is provided of a P18T and P18T-KRASG12D CRC organoid during and after afatinib (dual EGFR/HER2 inhibitor) therapy. Green, alive nuclei. Red, marked nuclear remnants of dead cells. Color code legend is provided at the bottom of panel C. (C) Patient-derived CRC organoids P8T and P26T were treated and analyzed as described in B. In general, cancer cells that survived drug therapy rapidly reignited cell proliferation after drug release. veh, vehicle (DMSO); sel, selumetinib; afa, afatinib; afa+sel, afatinib+ selumetinib; SCH, SCH772984; SCH+sel, SCH772984+selumetinib.