Real-Time Apoptosis and Necrosis Detection in 3D Spheroid Cell Models

Part # PS346


Andrew L. Niles, Kevin R. Kupcho, Terry L. Riss, Dan F. Lazar and James J. Cali
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

Cancer spheroids grown in vitro often share many of the same structural and biological complexities of native tumors and have become a convenient means to approximate the relative efficacies of new chemical entities. In this work, we examined the pharmacodynamics of apoptosis induction in spheroids using 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 bortezomib, paclitaxel, or panobinostat to detail the response magnitude, potency and kinetics resulting from continuous exposure. Luminescent and fluorescent data were continuously collected every 30 minutes during a 48h exposure using a conventional multimode plate reader equipped with atmospheric control. After the exposure period, the spheroids were imaged by fluorescence microscopy to assess necrotic burden as well as assayed for remaining cellular viability using ATP content. All measures were compared to untreated control spheroids. 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 efficacies of new chemical entities with respect to their capability to penetrate and induce apoptosis in spheroids and/or other more complex multicellular models.

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