The assessment of autophagy in tissues is mostly based on the detection of autophagosomes using transmission electron microscopy (TEM),14,15
a technique that is laborious and requires considerable expertise, especially at the level of image analysis. Thus, various cytoplasmic structures have been misidentified as autophagic vacuoles in the recent literature, most likely because of the lack of morphological expertise to correctly interpret TEM images.7
Moreover, artifacts can be generated during fixation and staining procedures, and other organelles such as the endoplasmic reticulum can swell in stressed or dying cells, leading to an autophagy-unrelated cytoplasmic vacuolization.7
Immunoelectron microscopy using antibodies against autophagosomal proteins can improve the detection of autophagosomes,8,16
but this method is particularly cumbersome. In preclinical models, quantification of GFP-LC3 puncta by fluorescence microscopy in GFP-LC3-transfected cells and the immunoblotting-based detection of the conversion of LC3-I to LC3-II have been widely used.2,7,17,18
However, despite numerous preclinical studies suggesting that the pharmacological modulation of autophagy might be useful in cancer treatment,13,19-22
the clinical implications of elevated or suppressed autophagy in human malignancies remains largely unknown. Hence, a convenient method for detecting endogenous LC3 puncta that is applicable to paraffin-embedded human tissues may constitute a decisive advantage.
In mammals, LC3 is expressed as 3 isoforms: A, B and C. Here, we used an antibody that was generated against the N-terminus of human LC3B to investigate the expression and subcellular localization of LC3B in human or mouse cancer cells. We gave preference to LC3B due to its broad tissue specificity and its previous characterization as an autophagosome marker in cancer.7,23,24
Nonetheless, to obtain a complete picture of the formation of autophagic vacuoles in tissues, it might be important to establish similar protocols as the one described herein for the detection of additional LC3 isoforms or more distant members of the LC3 family (such as the γ-aminobutyric acid receptor-associated protein, GABARAP, and the Golgi-associated ATPase enhancer of 16 kDa, GATE-16).
Relatively little is known about autophagy levels in healthy (or at least nonmalignant) human tissues. In our study, many non-neoplastic cells, notably colon and placental cells, harbored a weak LC3 staining, confirming previous studies on these organs.25-27
In kidneys, we observed a diffuse staining in the epithelial lining of tubules and glands, consistent with the physiological role of autophagy in renal function.28
Although tumor cells tended to stain more intensely for LC3 than normal cells, it should be noted that normal immune cells, stromal fibroblasts or tumor infiltrating lymphocytes (TILs) could be strongly positive for LC3, rendering it difficult to distinguish normal stromal cells from cancer cells with a fibroblastoid or lymphoid morphology. Thus, LC3 immunostaining cannot be used as the sole criterion to distinguish malignant from non-malignant cells. As it was previously described for colorectal cancer,26
we found that breast tumor cells tend to express higher LC3 levels than the adjacent, nonmalignant parenchyma. Moreover, LC3 puncta were generally absent from nontransformed breast epithelia. These staining patterns were comparable to those previously described in breast carcinoma.29
However, the prognostic or predictive value of LC3 expression remains controversial13,29,30
and requires further studies on large patient cohorts.