Abstract 4296: A real-time annexin V method for monitoring programmed cell death
Authors: Kupcho Kevin , Niles Andrew , Shultz John , Grailer Jamison , Zhou Wenhui , Hurst Robin , Hartnett Jim , Riss Terry , Lazar Dan , Cali James 
Published: July 2017

Abstract
Efficacious and durable anti-cancer responses are driven by both the selective death of malignant cells and the induction of immunostimulatory activities. Limited but promising clinical evidence suggests that provocation of an unknown balance of inflammatory- and non-inflammatory cell death may be key for orchestrating these positive outcomes. Therefore, identifying and characterizing new clinically useful small molecules and biologic inducers (or combinations thereof) which promote a spectrum of programmed cell death in in vitro screening environments remains critically important. Unfortunately, current screening methods are either insufficiently robust, cost- or resource-prohibitive, or provide no means for initial characterization of the kinetics of programmed cell death. To address this unmet need, we developed a real-time, live cell assay method that utilizes a fully homogeneous, bioluminescent annexin V reagent. The method does not require laborious washing and sample preparation steps associated with traditional annexin methods and is fully compatible with plate-based multimodal signal detection systems. The system contains two annexin proteins which have been engineered to contain separate and distinct complementing domains of a binary luciferase. Additionally, the system contains a novel time-released luciferase substrate and a cell impermeable, fluorogenic DNA dye for monitoring necrosis. Because the annexin-luciferase fusion pairs have only modest affinity for each other, luminescence remains low until phosphatidylserine exposure, a hallmark of the programmed cell death phenotype, brings annexin monomers into close proximity facilitating complementation of the luciferase sensor. The assay reagent can be applied at dosing for real-time measurement of the dose-dependency and magnitude of programmed cell death progression. This work describes our efforts to characterize and validate the performance of the bioluminescent annexin assay using relevant cell death induction models. First, the assay was shown to be functionally concordant with a flow cytometry annexin method in a dose-response model with bortezomib at exposure periods known to produce both early and late-apoptosis (with necrosis) phenotypes. Next, we assessed the performance of the assay using a limited training set of small molecule and biologic inducers of programmed cell death that utilize different mechanisms of action (i.e., apoptosis and necroptosis) and by using a number of diverse but representative cancer cell lines. We conclude that the bioluminescent annexin method provides a new kinetic approach to efficient and effective detection of programmed cell death mechanisms in real time in a convenient homogeneous format.
Citation Format: Kevin Kupcho, Andrew Niles, John Shultz, Jamison Grailer, Wenhui Zhou, Robin Hurst, Jim Hartnett, Terry Riss, Dan Lazar, James Cali. A real-time annexin V method for monitoring programmed cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2017-4296