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Abstract Phenotypic screening is a valuable tool for analysis of the complex mix of effects that drug treatment has on cells. Traditionally image-based screening uses fixed cells stained with reagents at a pre-chosen end point, however our technique combines kinetic, label-free readouts with non-perturbing fluorescence reporters to enable information-rich quantification of compound effects. A549 lung cancer cells were treated with a library of over 800 FDA-approved drugs while images were acquired in the Incucyte® Live-Cell Analysis System every two hours for four days. Cell growth and viability were measured using label-free readouts of confluence and dead cell count while cell cycle, cell death and Akt pathways were investigated using multiplexed fluorescent readouts. Cytotoxicity was measured using label-free Incucyte® AI Cell Health Analysis Software Module which uses two neural networks to segment individual cells and classify them as live or dead. Assay robustness was quantified using the time course of positive and negative controls (camptothecin and vehicle, respectively). Z’ reached a maximal value 0. 9 between 48h and 72h; at 72h 4. 4% of the compounds within the library induced 50% cell death. Correlation between % Live cells and % Confluence indicated 3 types of effect: cytotoxic (low viability, low confluence), cytostatic (high viability, low confluence), and none (high viability, high confluence). Morphological changes were observed in a multitude of wells with 29 compounds inducing cell enlargement 1800 µm2, suggesting induction of senescence. Hits from fluorescent readouts were identified as the compounds which perturbed the quantified readout more than 3 standard deviations away from the mean value of the vehicle. Mechanisms of cell death were examined by correlative analysis of the total dead cell population (label-free cell death identification), caspase-active (caspase 3/7-reagent positive) and Annexin V positive cells. This enabled rapid identification of compounds which induced apoptosis by caspase-independent pathways, including the serotonin-selective reuptake inhibitor Sertraline. Kinetic plots of cell cycle stage (% S|G2|M or G1) revealed contrasting mechanisms. Mycophenolic acid induced cell cycle arrest, observed as a consistent increase in the percentage of cells in G1 over time. However flumazenil displayed repeated, transient peaks in the percentage of cells in S|G2|M over the same 72h period, indicating synchronisation behavior. Incucyte® Live-Cell Analysis System as a phenotypic screening platform provides valuable kinetic data from cells within the physiologically relevant environment of a standard incubator. Label-free readouts yield direct cellular information and can be combined with fluorescent data to provide vastly more information than a single endpoint readout. Citation Format: Kirsty McBain, Gillian Lovell, Jasmine Trigg, Nicola Bevan, Daniel Appledorn. Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4722.
McBain et al. (Fri,) studied this question.