Measuring Autophagy in the Context of Cancer

Autophagy plays multiple roles in the formation and progression of cancer, including both suppressive and promotive roles. It not only impacts cancer cell growth and viability directly but also has a significant role through its effects on the tumor microenvironment. Measurement of autophagy can be confusing and sometimes misleading due to the inherent difficulty of measuring both the formation and turnover of molecules involved in the autophagic process. The LC3 proteins serve as autophagosomal markers and are the basis for most of the assays used for measuring autophagy. Since each of the current assays for autophagy has significant limitations, the use of multiple assays for the analysis of autophagy in most contexts is highly advised. Here we outline three assays that are commonly used to evaluate autophagic flux in cells. These assays include the determination of LC3II formation and LC3II and p62 turnover by use of Western Blotting, quantification of LC3 puncta, and the measurement of autophagic flux using tandem labeled mCherry-GFP-LC3.