Specific localization of LC3B in autophagosome : a correlative labelling study with nanoparticle in oral squamous cell carcinoma

Autophagy, a mechanism of cellular self-consumption for recyclingof intracellular components, has recently attracted much interest in cancer therapy research. This is because autophagy inhibition is reported to increase sensitivity to radiotherapy and chemotherapy in various cancers (Lai et al., 2014). Autophagy activity was first described by electron microscopy (EM) and the latter remains one of the standard methods to study autophagy activity because its components in different stages can be identified by their distinctive ultrastructures, in particular autophagosome, which occur at an early stage of autophagy process (Mizushima et al., 2002; Eskelinen et al., 2011; Martinet et al., 2014). During autophagy, intracellular materials are sequestered by phagophores, turning into autophagosome and fused with lysosome for degradation (Lai et al., 2014; Mizushima, 2004). Under EM examination, the autophagosome is characterized by its double membrane limited spherical structure (3, 4), however, such characteristic is also shown by swollen mitochondria, a common feature of tumor cells. This complicates the identification of autophagosomes in tumor cells (Martinet et al., 2014; Ghadially, 1985). An inaccurate identification of autophagosome would hinder result interpretation in therapeutic research such as assessing the impact of autophagy in drug resistance as well as testing the potency of a developmental autophagy inhibitor that aims to increase sensitivity to chemotherapeutic agents. Taken together, a more refined autophagosome identification method at ultrastructural level is needed. Microtubule-associated protein light chains 3 (LC3) is a specific marker for the autophagosome and consists of three main members including LC3A, LC3B and LC3C (He et al., 2003). LC3B is regarded as a better autophagosome marker and more importantly its expression pattern has been previously described in oral SCC tissue (Girolamo et al., 2013; Liu et al., 2014). LC3B, in combination with our established correlative immunelabelling method (Killingsworth et al., 2012), holds the potential to constitute a useful method to identify autophagosomes precisely at the ultrastructural level and thus provides a powerful approach for assessment of autophagy in cancer therapy research. This study investigated the specificity of LC3B antibody in the autophagosome using correlative immunolabelling conjugated with quantum dots (QDs).