Abstract 1834: Real-time visualization and quantification of flexible, multi-parameter indicators of cancer cell heath and metabolism using live-cell analysis
Authors: Schramm Cicely L. , Rauch John N. , Oupicka Libuse , Skerlos Laura A. , Bevan Nicola J. , Lovell Gillian F. , Perez-Garrido Sandra , Filonov Grigory S. , Chen Yong X. , Kovalenko Ilya , Foltin Susan , Campwala Hinnah , Dale Timothy J. , Appledorn Daniel M. 
Published: September 2020

Abstract
The ability to perform concurrent, dynamic measurements of multiple parameters of tumor cell health can provide valuable mechanistic insight. For example, a single compound may exhibit cytotoxic and/or cytostatic properties, depending on the tumor cell type being targeted as well as concentration and duration of treatment. In addition, evaluating immune-cancer cell interactions or metabolic exchange between cancer and stromal cells can be a critical component of elucidating mechanism of action. However, standard methods of evaluating compound effects on tumor cell health are largely limited to single parameter, population-based, endpoint measurements. Here we describe a new live-cell imaging solution providing flexibility in fluorescent readouts of cancer cell health, metabolism, and cell-cell interactions. Phase and fluorescent images were acquired and analyzed using an IncuCyte live cell analysis system. Changeable filter sets enabled selection of fluorescent readouts in designated combinations of green, orange, red, and/or NIR spectra as well as a dual excitation, single emission filter set to provide ratiometric measurements of cytoplasmic ATP. Using this approach, we demonstrate concentration- and time-dependent effects of compounds on cell cycle and cell death. Kazusamycin A (0.08-20 nM) treatment of HT-1080 cells resulted in a concentration-dependent cell cycle arrest as measured by a fluorescence ubiquitination-based cell cycle indicator. An increase in the percentage of cells in G1 (22% vs 31% of vehicle or kazusamycin A-treated cells, respectively) was observed as early as four hours after treatment and increased over the 3-day time course. Cell death was evaluated concurrently using an Annexin V reagent. Apoptosis was observed only at high concentrations (6.67-20 nM) and after 18 hours of incubation. Similar results were observed in MDA-MB-231 cells. Incorporation of genetically encoded and/or live-cell immunocytochemistry reagents enables monitoring subpopulations of cells in co-culture models. We utilized these techniques to monitor dynamic readouts in target and effector populations in an immune cell killing assay. MDA-MB-231 cells were transduced to express a fluorescent nuclear marker and were co-cultured with PBMCs with and without activation (ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator + IL-2). A decrease in nuclear counts accompanied by apoptosis (Annexin V-positive cells) of MDA-MB-231 target cells was observed within 27 hours in the presence of activated PBMCs. Measurements of target cell proliferation and apoptosis were unaffected by the presence of PBMCs without activation cocktail. Additional example datasets of cancer cell metabolism, proliferation, and death readouts in mono- and co-culture models will be shared to illustrate the value of flexible, multi-parameter live cell analysis.
Citation Format: Cicely L. Schramm, John N. Rauch, Libuse Oupicka, Laura A. Skerlos, Nicola J. Bevan, Gillian F. Lovell, Sandra Perez-Garrido, Grigory S. Filonov, Yong X. Chen, Ilya Kovalenko, Susan Foltin, Hinnah Campwala, Timothy J. Dale, Daniel M. Appledorn. Real-time visualization and quantification of flexible, multi-parameter indicators of cancer cell heath and metabolism using live-cell analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1834.