In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

Autophagy inhibitors are considered as potent chemosensitizers and might therefore be introduced into combination regimens for optimal anticancer therapies. Methods to measure autophagy include electron microscopy for quantifying autophagosomes, immunoblotting for detecting the lipidation of microtubules-associated protein 1 light chain 3 (also known as LC3), or immunofluorescence microscopy for monitoring the redistribution of LC3 from a diffuse pattern to a punctate one. 41 Complementary techniques are necessary as the autophagosomal accumulation of LC3 can be influenced by lysosomal acidification. For instance, another way is to assess the autophagic flux, thanks to several methods: pulse-chase, immunoblotting, or luciferase-based methods for monitoring the degradation of autophagic substrates. A luciferase coupled with LC3 has recently been used to develop a real-time HTScompatible assay for monitoring the autophagic flux. Moreover, firefly luciferase has been shown to constitute a preferential autophagic substrate and is hence a trustful marker of autophagic degradation that may be applicable to HTS studies. In addition, recent developed assays allow determining the phosphorylation status of substrates from critical autophagy regulator kinases, such as mTORC1 and UNC51-like kinase 1. Besides, several of these assays have been recently adapted for HTS. Finally, these methods should be used to identify the underlying molecular mechanisms of autophagy more than quantifying its levels. [42] [43] [44] [45] Detection of necrosis, ferroptosis, and immunogenic cell death