Methods to measure autophagy in cancer metabolism

Abstract

Autophagy, a dynamic pathway in which intracellular membrane structures sequester portions of the cytosol for degradation, plays multiple roles in physiological and pathological processes. Autophagy may have suppressive and promotive roles in the formation and progression of cancer. A growing number of methods to identify, quantify, and manipulate autophagy have been developed. Because most of these methods are semiquantitative and have significant limitations, it is important to emphasize that a combination of these assays is recommended for the analysis of autophagy. Here, I briefly discuss the autophagic process, its role in disease, and I summarize some of the best-known and most widely used methods to study autophagy in vitro in the context of cancer, including transmission electron microscopy (TEM), detection and quantification of the autophagy protein LC3 by western blot, and the use of GFP-LC3 to quantify puncta by fluorescence microscopy and tandem labeled RFP/mCherry-GFP-LC3 fluorescence microscopy to measure autophagic flux.