The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing the assay response, and the signal is stable, allowing assay of multiple 96- or 384-well plates in the same experiment. In response to autophagic stimuli, including nutrient deprivation and various mTORC inhibitors (e.g., PP242 and rapamycin), autophagic degradation of expressed LC3 reporter was evident by reduced assay signal. In contrast, in response to both upstream (e.g., 3-MA and wortmannin) and downstream (e.g., bafilomycin A1 and chloroquine) inhibitors of the autophagy pathway, degradation of the autophagic reporter was effectively blocked and assay signal was consistently increased as predicted. Compound effects were time dependent and stratified according to expected potency and efficacy of the test agents employed. The use of a mutant reporter based on LC3G120A further demonstrated the specificity of the wild-type LC3 reporter for the detection of autophagic activity. When assayed in 384-well plates with automation, HEK293 autophagy reporter cells produced Z’ values of ~0.7 in response to autophagy induction with PP242, while subsequent blockade of autophagy with bafilomycin A1 resulted in Z’ values of ~0.8. This data, and subsequent LOPAC library screening, indicates the potential utility of this assay method for HTS applications. In addition, the HEK293 autophagy reporter cells can be induced to form 3D cell spheroids, thus allowing investigation of assay performance in this more complex model. Autophagy reporter levels increased with increasing spheroid size (up to 650 μm diameter tested) in a manner proportional to a surrogate measure of viable cell number. Importantly, both induction and inhibition of autophagic activity was easily detected following PP242 and bafilomycin A1 treatment, respectively. Using this novel plate-based assay system for the determination of autophagic flux, it is possible to screen test agents and quantitatively determine both the potency and efficacy of autophagy modulation.
Citation Format: Dan F. Lazar, Amani A. Gillette, Braeden L. Butler, Christopher T. Eggers, Brock F. Binkowski, Gediminas Vidugiris, Michael R. Slater, Dongping Ma, James J. Cali. A novel plate-based assay for screening autophagic activity in 2D and 3D cell culture models [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 3312. doi:10.1158/1538-7445.AM2017-3312