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

One of the best methods to study necrosis is the use of morphologic changes that can occur after death cell, thanks to light or electron microscopy. Indeed, the term necrosis can be defined as a mass of dead cells and does not depend from a specific cell death pathway. Specific characteristics of necrotic cells have been described, including nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of organelle function, and cytolysis through swelling, which leads to membrane break. 46 Nonetheless, if secondary necrosis represents the end point of all cell death modalities, primary necrosis, by contrast, constitutes an entire cell death mechanism with specific characteristics. It is difficult to identify specific markers of necroptosis as most of the biochemical processes that characterize necroptosis can also take place during apoptotic cell death, and the best way to detect necroptosis remains the analysis of a crucial kinase activation: receptor-interacting protein 1 (RIP1), which is on the crossroad of death signaling pathways. Its activation can be measured by enzymatic assay or by studying its phosphorylation on S161.The use of pharmacologic inhibitors such as Necrostatin-1 that inhibits RIP1 activity may be useful to identify necroptosis. 47 Another commonly used assay for necrosis detection measures the extracellular release of high-mobility group box 1 (HMGB1), a nonhistone chromatin-binding protein that normally regulates transcription, and kits are available based on enzymelinked immunosorbent assay tests for quantification of HMGB1. A recent biomarker for necrotic cells has also been described: peptidylprolyl isomerase A, also known as cyclophilin A (CYPA), seems to be specifically released from necroptotic cells. Enzyme-linked immunosorbent assay kits for the quantitative measurement of CYPA are also commercially available, and drugs that bind and inhibit CYPA are being explored and might be used to develop CYPA-oriented assays. Besides, CYPA-oriented assays and HMGB1 assays should be adapted to HTS assays for detecting necrotic cell death. At present, data concerning molecular mechanisms of necroptosis are scarce and require further research. 48, 49 Concerning the detection of ferroptosis, cells which undergo this form of cell death exhibit specific morphologic features such as smaller mitochondria with increased density that can be easily identified. Besides, the occurrence of ferroptosis can be confirmed using ferroptosis inhibitors such as Ferrostatin-1. 17 Finally, regarding immunogenic cell death, current research focuses on the development of inducers through vaccination assays with immunodeficient mice reconstituted with a human immune system and simultaneous detection of CALR exposure, ATP secretion, and HMGB1 release for assessing cell death. Guidelines for the detection of immunogenic cell death have recently been published. 50 Advantages and drawbacks of methods used for assessing cell viability, apoptosis, autophagy, and necrosis are presented in Table 1 .