Abstract 7528: Utilizing confocal live-cell imaging for evaluating therapeutic efficacy and toxicity in complex oncology models

Abstract Oncology research continues to progress towards utilizing more complex, translational cellular models to improve clinical outcomes. The need for complementary bioanalytical tools to enable clear insights from increasingly complex data has evolved in parallel. The Incucyte® CX3 enables multiplane, spinning disk confocal imaging of complex tumor models and organoids under physiologically relevant conditions. This poster will highlight the application of this technology, paired with integrated analysis tools, to evaluate the efficacy and off-target toxicity of therapeutic compounds. The effects of chemotherapeutic compounds were first evaluated in solid tumor models. For example, MCF7 cells stably expressing a nuclear-restricted green fluorescent protein were embedded in extracellular matrix and plated in the presence of a fluorescent dye to monitor apoptosis. Spheroid formation was monitored for 3 days, then the effects of camptothecin (0.3 - 10 µM) or cisplatin (3 - 100 µM) were tested. Automated analysis performed on confocal max projection images revealed a concentration-dependent decrease in spheroid growth and complementary increase in apoptosis. Many chemotherapeutic agents have been observed to induce hepatotoxicity in clinical cases. Therefore, the effects of compounds were also measured in a model of liver toxicity. Mouse hepatic organoids stably expressing a nuclear-restricted orange fluorescent protein were plated in a similar manner to cancer spheroids. Confocal multiplane images were acquired every 8 hours during formation (1 day) and for an additional four days following drug treatment. Camptothecin and cisplatin induced a concentration-dependent decrease in growth over time, with high concentrations (5-10 µM or 50-100 µM, respectively) inducing apoptosis. Importantly, fluorescent readouts of toxicity enabled by confocal imaging were more sensitive, as cellular debris can be difficult to distinguish from healthy organoids via brightfield readouts. Immune- and cancer-cell coculture models were also evaluated. SKOV-3 Nuclight Orange spheroids were seeded with a density range of activated (10 ng/mL CD3/CD28 for 72 hours) or non-activated PBMCs in the presence of Fabfluor-488-CD45 or Fabfluour-488-IgG1 and optigreen background suppressor. Co-cultures were imaged over 6 days using confocal multiplane acquisition. The results showed a density-dependent decrease in orange area with increasing PBMC density for activated PBMCs, indicating target cell death. Additionally, following immune-mediated tumor killing, we observed a density-depending increase in green area with PBMCs expansion. Taken together, these data highlight the ability of multiparameter, multiplane confocal live cell analysis to provide clear, rapid insights from a variety of complex pre-clinical models, including immuno-oncology and organoid-based toxicity readouts. Citation Format: John Rauch, Jasmine Trigg, Kirsty McBain, Jonathan Bezenah, Libuse Oupicka, Richard Lister, Laura Skerlos. Utilizing confocal live-cell imaging for evaluating therapeutic efficacy and toxicity in complex oncology models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7528.