The Development of an Immunohistochemical Method to Detect the Autophagy-Associated Protein LC3-II in Human Tumor Xenografts

Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.