Fig. 1

Unbiased morphological profiling of anticancer agents using cell painting assay. (a) An overview of the strategy for the screening procedure is illustrated. Human osteosarcoma U2OS cells were treated with a custom-built anticancer library at 3 µM, while cisplatin was used at 300 µM and oxaliplatin at 500 µM, supplemented with a selection of agents inhibiting transcription (i.e. 3 µM epirubicin, 3 µM idarubicin, 10 µM abemaciclib, 1 µM becatecarin, 1 µM trabectedin, 5 µM BMH21, 1 µM CX5461, 15 µM aclarubicin, 1 µM lurbinectedin, 50 µM topotecan, 20 µM metarrestin, 200 µM DRB, 1 µM flavopiridol, 1 µM triptolide) or left untreated (CTR), for 4 h. Subcellular organelles were stained according to the cell painting assay and cells were subjected to high-content microscopy, image segmentation and subsequent cell features extraction. After sub-setting two groups from the dataset, i.e. transcription inhibitors and untreated (CTR) group, a random forest (RF) binary classifier was trained, and feature importances were thereafter extracted from the latter. (b) Representative fluorescence and transmitted light (TL)-based images show the panel of acquired channels, i.e. Hoechst 33342 (nucleus, DNA; DAPI channel), MitoTracker Deep Red (mitochondria; Cy5 channel), concanavalin A (endoplasmic reticulum; FITC channel), SYTO 14 (nucleoli, cytoplasmic RNA; Cy3 channel), phalloidin (actin; Texas Red channel) and TL (brightfield), for a selection of agents. Scale bar equals 10 μm. (c) The displayed relative Gini index decrease represents the importance of features for classification. Colors represent the acquisition channels from which the feature was extracted. (d) Hierarchical cluster dendrogram based on the weighted distance matrix calculated using Gower’s similarity coefficient identifies a grouping pattern of the evaluated chemical compounds. Agents from the custom-built anticancer library are depicted in blue, supplemental transcription inhibitors in rose red, anticancer transcription inhibitors in brown and an adjacent green dot indicates confirmed immunogenic cell death (ICD) inducers