Abstract 710: Real-time apoptosis and necrosis detection in 3D spheroid cellmodels

Abstract

Abstract The structural and biological complexity of cancer spheroids make them uniquely suited to address or approximate the pharmacodynamics of new chemical entities directed at chemotherapeutic reduction of tumor masses. Unfortunately, this same complexity represents a substantial technical challenge for measuring apoptosis and necrosis using conventional methods in real time. To address this challenge, we explored the use of a multiplexed bioluminescent and fluorescent, real-time assay reagent which utilizes annexin V fusion proteins containing binary elements of a complementing luciferase, a time-released luciferase substrate and an excludable DNA membrane integrity dye. The assay reagent was added to HCT-116 and HepG2 spheroids dosed with serial dilutions of panobinostat, bortezomib, or paclitaxel to examine the magnitude, potency and kinetics of the responses. Luminescent and fluorescent data were continuously collected throughout the 48h exposure using a conventional multimode plate reader equipped with atmospheric control. After the exposure period, the treated spheroids were imaged by fluorescence microscopy to assess necrotic burden as well as analyzed for remaining cellular viability using ATP content compared to untreated control. Consistent with their known modes of cytotoxic action, bortezomib produced the first measurable phoshatidylserine (PS) exposure (~8h), followed by panobinostat (~12h) and paclitaxel (18-22h). The apoptotic potencies and magnitudes of response matured thereafter as a function of time until 48h. Secondary necrosis resulting from completion of the apoptotic program lagged PS exposure in both time and magnitude. Imaging results for necrosis and ATP determinations provided orthogonal and inversely complementary verification of data values obtained with the apoptosis assay reagent. Taken together, this workflow may help define the in vitro pharmacological disposition of new chemical entities with respect to their capability to penetrate and induce apoptosis in spheroids and/or other more complex multicellular models. Citation Format: Andrew L. Niles, Kevin R. Kupcho, Terry L. Riss, Dan F. Lazar, James J. Cali. Real-time apoptosis and necrosis detection in 3D spheroid cellmodels [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 710.