LB13
The development of an immunohistochemical method to detect the autophagy associated protein LC3-II, in human tumour xenografts
Sarah Holt, Beata Cieniuch, Kevin Randall, Dominic James, Martin Kelly, Robert Wilkinson
AstraZeneca Pharmaceuticals, Cheshire, UK
In recent years a large number of oncology drugs have entered clinical trials, which in addition to their known anti-proliferative and/or pro-apoptotic effects, may induce autophagy. Consequently this has led to a demand for biomarkers of autophagy to evaluate this process initially in pre-clinical models and ultimately in the clinic. The field of autophagy is still evolving and there is a constantly changing criteria for the assessment of the process in cells, tissues and organs (Klinosky, et al., 2008). The gold standard technique for analysing autophagy in mammalian cells remains electron microscopy (EM), which has limitations in that it is labour intensive, only a small sample set can be analysed at any one time and it is often difficult to do on human tumour xenograft tissue samples.
We have therefore taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagasome associated protein microtubule-associated protein 1 light chain 3 (LC3), in human tumour xenografts. After synthesis, the C-terminal region of LC3 is cleaved to form LC3-I. When autophagy is induced, some LC3-I is converted into LC3-II, which is tightly bound to the membrane of the autophagasome. It is thought that detection of endogenous LC3-II by IHC could be difficult due to a lack of abundance (Martinet, et al., 2006). In order to enrich our samples for LC3-II we used chloroquine to increase the number of autophagasomes. Analysis of a human glioblastoma tumour xenografts (U87MG) derived from mice treated with/without chloroquine, demonstrated the detection of LC3-II puncta in histological tissue sections. It is hoped that this method can be extended to other preclinical tumour models in order to assess autophagy following treatment with novel anti-cancer therapies.
