Most Autophagic Cell Death Studies Lack Evidence of Causality

Abstract

Autophagy plays a critical role in maintaining cellular homeostasis and is implicated in various physiological and pathological processes, including cancer, neurodegeneration, and metabolic disorders. Although typically associated with cell survival, autophagy has also been proposed to contribute to cell death, referred to as autophagic cell death (ACD). However, the identification of ACD remains contentious due to inconsistencies in experimental methodologies and terminological misuse. In this study, we systematically evaluated 104 research articles published in 2022 that claimed to demonstrate ACD. Articles were assessed based on established criteria, including evidence for autophagy, evidence for cell death, exclusion of apoptosis, and experimental designs demonstrating causality. Our findings reveal that only 12.5% of the articles fulfilled all ACD criteria, while 37.5% provided only correlation-level evidence. Additionally, 54.81% failed to demonstrate autophagy flux, 32.7% relied on viability loss rather than direct evidence of cell death, and 45.0% of studies utilizing autophagy inhibition failed to demonstrate actual inhibition of autophagy. Inconsistent terminology was also prevalent, with autophagy-mediated cell death often misclassified as ACD and ACD frequently misused to describe autophagy co-occurring with cell death. These issues highlight a lack of rigor in current practices, with correlation-level evidence, inappropriate experimental designs, and terminological misuse undermining study robustness. To address these challenges, we developed a systematic workflow providing experimental and analytical guidance for classifying evidence for different modes of autophagy. Our analysis underscores the need for greater rigor, standardized approaches, and precise terminology to advance understanding of the interplay between autophagy and cell death.