Abstract 5337: Homogeneous bioluminescent immunoassay for hKi-67 provides a simple and robust screening tool for antiproliferative agents

Abstract We present a robust, homogeneous, and luminescent microplate-based immunoassay utilizing Lumit® technology for fast and quantitative detection of human Ki-67, a marker of cell proliferation. This assay enables efficient screening of antiproliferative agents, demonstrating exceptional performance in assessing antiproliferative activity across multiple mechanisms of action. We evaluated the Lumit® hKi-67 immunoassay in HCT116 colorectal cancer cells treated with increasing concentrations of four agents known to inhibit proliferation via different mechanisms: palbociclib (CDK4/6 inhibitor), samuraciclib (CDK7 inhibitor), nutlin-3A (MDM2/p53 interaction inhibitor), and thymidine (dNTP pool perturbation). With all four test agents, the Lumit® hKi-67 immunoassay detected a marked, dose-dependent 63-82% reduction in Ki-67 after only 24 hours, and an even greater 79-98% reduction at 48 hours, demonstrating a fast and reliable measure of antiproliferative effects. A same-well multiplex with a cell-impermeant fluorogenic DNA dye demonstrated no cell death provoked by these compounds at the doses and times tested. In contrast to the large decrease in the proliferation marker Ki-67 elicited within 24 h of treatment by these antiproliferative agents, the response windows achieved with assays of viable cell number were significantly smaller at the early time point. Only 25-40% less viable-cell DNA was observed after 24 h compound treatment vs. untreated controls, with 48 h required to produce response windows of 57-89%. Similar observations were obtained with two other viability assays, one detecting ATP levels and the other live-cell protease activity. However, ATP levels were reduced more than expected by 24 h treatment with thymidine, likely due to perturbation of metabolic activity. Equally concerning, at 48 h, the live-cell protease assay and, especially, detection of ATP levels dramatically underreported the decrease in viable cell number (relative to time-matched untreated controls) in response to palbociclib and nutlin-3A. This latter effect is likely due to the substantial increase in cell size induced by palbociclib and nutlin-3A even as they inhibit cell proliferation. The strong antiproliferative effects induced by palbociclib and nutlin-3A and detected with the hKi-67 immunoassay, however, were further validated by two additional assays: Ki-67 immunofluorescence and BrdU ELISA. Notably, the Lumit® assay provides faster, more robust data compared to these more labor-intensive alternatives. This study highlights the Lumit® hKi-67 immunoassay as an attractive, high-throughput alternative for screening antiproliferative agents, offering a simpler, faster, and more consistent approach than traditional methods. It is poised to be an invaluable tool in drug discovery and preclinical screening. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. Homogeneous bioluminescent immunoassay for hKi-67 provides a simple and robust screening tool for antiproliferative agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5337.