Quantitative analysis of autophagy-related protein LC3B by quantum-dot-based molecular imaging

Abstract

Autophagy is a process that facilitates the maintenance of intracellular homeostasis by removing unnecessary or dysfunctional cellular components. It plays a role in inhibiting tumorigenesis in the early stage of the disease and might promote progression after tumor formation. Microtubule-associated protein light chain 3 (MAPLC3, better known as LC3), isoform B (LC3B), is one of the most commonly used markers of autophagy. The expression of LC3B has been studied in many cancers and was shown to be closely related to tumor progression. Here, we provide detailed experimental steps for the quantitative detection of LC3B expression in cancer tissue by quantum-dot-based molecular imaging. As compared to the traditional immunohistochemistry (IHC) employing standard fluorochromes, the present method has a higher signal amplitude and improved sensitivity enabling the accurate quantitative detection, which provides a foundation for functional research and the clinical application of LC3B biomarker.

Introduction

Autophagy maintains intracellular homeostasis by eliminating aged or damaged organelles, misfolded proteins as well as pathogens through lysosomal degradation. The degradation products (such as fatty acids and peptide fragments) are eventually transported to the cytoplasm for further metabolic processing such as energy generation or recycling (Yang & Klionsky, 2020). Autophagy plays a complex role in tumor development. On the one hand, autophagy can inhibit tumor formation by maintaining chromosome homeostasis and preventing local inflammation. Hence, it is considered a tumor suppressor during early stages. On the other hand, autophagy provides the tumor a survival advantage by enhancing its drug resistance after tumor formation (Gewirtz, 2020). Consistently, studies have shown that the anti-tumor effect of anti-cancer agent could be enhanced by inhibiting autophagy (Liu et al., 2020; Nazim, Yin, & Park, 2020).

Microtubule-associated protein light chain 3 (MAPLC3, better known as LC3), a mammalian homolog of yeast autophagy-associated protein 8 (Atg8), has three isoforms (LC3A, LC3B and LC3C). Among them, LC3B is one of the most commonly used markers of autophagy (He et al., 2014). Each LC3 isoform has two forms, autophagy-inactive cytosolic LC3-I and autophagy-active processed LC3-II. During autophagy, LC3-II is generated by lipidation of LC3-I through an ubiquitin-like conjugation system. Consequently, the amount of LC3-II is correlated with the level of autophagy and can be used as a marker of autophagy (Schaaf, Keulers, Vooijs, & Rouschop, 2016). In cancer, LC3B is the most commonly detected subtype of LC3, and its antibody has higher reactivity with the LC3-II form rather than LC3-I. The expression of LC3B is widely studied in many cancers including breast, pancreatic, colon, thyroid, lung, ovarian, hepatocellular cancer and melanoma (Lazova et al., 2012; Liang et al., 2020; Xu, Yang, & Li, 2020). LC3B expression encompassed both the punctate and diffuse compartments of the cytoplasm in solid tumors, and the punctate LC3B staining could reflect the existence of autophagosomes and has been proved to be closely related to tumor progression (Lazova et al., 2012).

Traditional immunohistochemistry (IHC) is currently the main method for detecting LC3B in traditional formalin-fixed, paraffin-embedded (FFPE) tissues, but it can only perform semi-quantitative detection of LC3B. Quantum dots (QDs) are semiconductor nanocrystal with characteristics including high fluorescence intensity, stable fluorescence signal, good histocompatibility and multi-molecule coaxial imaging. These characteristics make it widely used in the field of biomedicine. Compared to traditional IHC, QD-based molecular pathology for molecular targeted imaging has better signal strength and higher sensitivity, enabling accurate quantitative detection (Pleskova, Mikheeva, & Gornostaeva, 2018). Here, we provide detailed experimental steps for the quantitative detection of LC3B expression in cancer tissue by QD-based molecular imaging.