Abstract 1428: Luminescent immunoassays for homogeneous determination of cell proliferation and senescence

Abstract

Abstract We have developed homogeneous, luminescent immunoassays for simple and rapid determination of Ki-67 and uPAR levels in 96-well format, thereby enabling efficient and quantitative assessment of proliferation and senescence in mammalian cell culture. Assessment of proliferative capacity is of key importance in numerous research areas, including cancer and various proliferative disorders, assessment of cell fitness (e.g., T cell fitness, stem cell characterization), and evaluation of test compound or drug effects. Cell senescence, characterized in part by loss of proliferative capacity, is an altered, proinflammatory state of cells induced in response to developmental ques, aging, disease, or acute stressors typically involving DNA damage. Ki-67 is a nuclear protein widely used as a marker of proliferation due to its pervasive expression in cycling cells but absence from non-dividing (G0) cells, whether quiescent, senescent, or terminally differentiated. In contrast, urokinase plasminogen activator receptor (uPAR) is a cell-surface protein whose expression was recently reported to increase significantly with the induction of senescence. Of note, commonly used assays of Ki-67 and uPAR levels typically rely upon non-homogeneous methods, including imaging, flow cytometry, or western blotting. Application of Lumit™ technology enabled our development of no-transfer, no-wash immunoassays for Ki-67 (lytic) and uPAR (nonlytic) determinations directly in cell wells. Treatment of human CD8+ T cells with CD3/CD28 T cell activator produced more than a 10-fold upregulation of Ki-67 levels, consistent with induction of T cell proliferation. In contrast, treatment of HCT116 colorectal cancer cells for 48 h with the antiproliferative compounds doxorubicin (100 nM) and nutlin-3A (10 μM) produced 65% and 90% reductions, respectively, in Ki-67 levels normalized to a measure of viable cell number, consistent with their relative antiproliferative activities. Dose-dependent treatment of HCT116 cells with nutlin-3A for 24 and 48 h produced 69% and 93% reduction, respectively, in normalized Ki-67 levels with a potency of ~2.5 μM. Treatment of HCT116 and A549 cancer cell lines for 72 h with 10-20 μM nutlin-3A, in addition to causing profound reduction in Ki-67 levels and loss of proliferative capacity, produced 2.3- to 4-fold increases in cell-surface uPAR levels, reflective of the emergence of the senescence phenotype. These luminescent, homogeneous Ki-67 and uPAR immunoassays provide an efficient means to quantitatively assess changes in cell proliferation and senescence in cell culture models for basic research and drug discovery applications. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. Luminescent immunoassays for homogeneous determination of cell proliferation and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1428.