Abstract Live cell imaging assays are essential for in vitro cancer biology, enabling interrogation of cellular state over time, tracking changes in growth and phenotype. High content imaging (HCI), where resolution and multiparametric quantification is key to extracting insight from cellular features, is stymied by slow imaging times, preventing live cell screens at scale. However, the Araceli Endeavor® newly enables live cell high throughput high content imaging, capturing the majority of cells in a 1536-well plate in 6 minutes in 5 channels with submicron resolution. Here, we apply this speed to cancer biology, scaling up the subcellular detail of HCI to live cell screening over a multi-day time-course. We first examine cell growth and quantify where cells are in the cell cycle based on DNA content and morphology, showing changes over time in response to drug treatment. Using fluorescent dyes, we further assess changes in cell death, mitochondrial function and cellular morphology, as well as evaluate cell proliferation. High resolution brightfield imaging, added to fluorescent imaging here in 30 seconds/run, provides a dynamic, unbiased look into off-target effects and generates the detailed image features needed for machine-learning-based analysis. Both proliferation and cell cycle measurements are traditionally done with fixed endpoint flow cytometry but these assays have limited insight. Live assays are far more revealing and vital to drug development. For instance, potential therapeutics may appear to suppress cancer in vitro, only for growth to rebound hours later, or this fixed endpoint may miss the window where a treatment is effective altogether. Unlike traditional HCI, Endeavor’s speed ensures that the first well is the same state as the last, not hours older. Overall, live cell HCI offers exquisitely detailed insight over time: here correlating cell growth, death, location, morphology, and mitochondrial dynamics in a single assay, at the speed and scale required for modern screening. Citation Format: Matthew Boisvert, Lydia Hernandez. A high content live cell cancer toolbox uniquely enabled by high throughput imaging 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 6411.
Boisvert et al. (Fri,) studied this question.