Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 374.