Fig. 4

Classification of drugs subtypes based on nuclear and toxicity features from a screening campaign. (a) Human osteosarcoma U2OS cells were pre-treated with a custom-made collection of 274 DNA/RNA-related drugs from TargetMol at 1µM, 10µM and 100µM or left untreated (CTR) for 2.5 h. Treatments pursued for 1.5 additional hours in the presence of 1mM 5-ethynyl uridine (EU). After fixation and nuclear counterstaining with Hoechst 33342, cells were permeabilized, EU was stained with an Alexa Fluor-488-coupled azide and cells were further stained with anti-Phospho-Histone H2A.X (Ser 139) antibody (γH2AX), followed by staining with Alexa Fluor 647 secondary antibody. To assess cell death U2OS cells were treated for 24 h, fixed and counterstained with Hoechst 33342. Images were acquired by fluorescence and transmitted light (TL) microscopy. Representative images of cells treated with 5 µM aclarubicin (ACLA), 10 µM etoposide (ETO), 300 µM cisplatin (CDDP), 1 µM dactinomycin (DACT), 500 µM oxaliplatin (OXA) or left untreated (CTR) are displayed. Scale bar equals 10 μm. A colormap representing confluency of cells is depicted for each condition. Scale bar represents 100 μm. (b) The clustered heatmap summarizes the fraction of maximum effects of each assessed parameter for 4 groups of interest (i.e. “ACLA-like”, “ETO-like”, cisplatin “CDDP-like” and doxorubicin “DOXO-like”) at a chosen concentration that maximized the overall effect of the assessed parameters at 4 h. Cell death was quantified via enumeration of Hoechst-stained nuclei, DNA damage activity via nuclear H2AX phosphorylation (γH2AX), inhibition of transcription via EU intensity in the nucleus and condensation of nucleoli (CON) was evaluated by a trained convolutional neural network (CNN) applied to nuclei patches from TL images. Color-codes indicating DNA interaction (DNA int.) type and enzymatic target (Enzym.) are reported for each condition. TOPO, topoisomerase; UNS, unspecific; POL, polymerase; LIG, ligase; GYR, gyrase. Adjacent green dots highlight confirmed immunogenic cell death (ICD) inducers while rose red dots indicates hits chosen for further validations. Data of each group are also reported in boxplots, CON in (c), EU in (d), γH2AX in (e) and cell death in (f). P-values were calculated using a pairwise Mann-Whitney test against the “DOXO-LIKE” group for each assessed parameter