Abstract 5266: Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis

Abstract

Abstract Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase-1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the IncuCyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the IncuCyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the IncuCyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements. Citation Format: Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn. Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5266.