A comparative and critical assessment of in vitro strategies for the detection and generation of anastatic cell populations

Late-stage Apoptosis, characterized by mitochondrial outer membrane permeabilization (MOMP) and Caspase activation, has traditionally been considered irreversible. However, recent studies describe "anastasis," a rare phenomenon in which cells recover from this seemingly irreversible state following stimulus removal. Yet, a central challenge in anastasis research is accurately describing the "point of no return," as specific apoptotic markers, such as cytochrome c release or Caspase activation, are dynamic rather than binary. Sublethal activation of these markers can occur early in the stress response, and recovery from this stage can lead to different outcomes, such as "failed Apoptosis." Therefore, accurately defining anastasis requires quantitative evaluation of apoptotic markers to distinguish it from Other recovery phenomena. Most studies rely on fluorescence-based Caspase biosensor's signal to track anastasis. Detection of the signal alone, without evaluating biosensor dynamics, can lead to misinterpretation of apoptotic stage. In this study, we assessed markers of late-stage Apoptosis to identify a reliable method to study anastasis. Using multiparametric time-lapse imaging, we demonstrate that the GFP-based caspase-3-like protease activity indicator (GC3AI) does not reliably indicate the late-stage Apoptosis. In contrast, we found that propidium iodide (PI) staining is a reliable and feasible method for generating anastatic cell populations. This work provides a framework for studying anastasis and thereby resolves methodological ambiguities and provides a strong foundation for studying anastasis.

Anastasis; Apoptosis; Caspase biosensor; Failed apoptosis; GC3AI.