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

Abstract

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.

Immunohistochemical analysis of macroautophagy

“…[3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] Detection guidelines have also been established for the correct use and interpretation of such methods. [19][20][21] Still demonstration of autophagy in tissue has not received extensive evaluation, particularly in clinical specimens for which genetic techniques (e.g., GFP-LC3 overexpression), autophagic flux measurements and injection of specific dyes are inconceivable.…”

“…Because mammalian LC3 is one of the most frequently used biomarkers for autophagy both in vitro 3,4,19,21 and in situ, [11][12][13][14][15][16] starved and nonstarved livers Nutrient deprivation induces autophagy in liver. To validate immunocytochemical methods for the detection of autophagy, nutrient deprivation was used as a trigger.…”

“…[9][10][11][12][13][14][15][16][17][18] Advantages of immunohistochemistry include a fast turnaround time (typically 2 d vs 5-7 d for TEM), low cost, ease of performance and widespread familiarity. However, most immunohistochemical staining protocols for autophagy-associated proteins have not been validated (e.g., by using autophagy-deficient tissue as negative control) or optimized to obtain high sensitivity.…”

Histologic changes of the fetal membranes after fetoscopic laser surgery for twin-twin transfusion syndrome

“…Autophagy activity can be detected by immunohistochemistry of autophagosome-associated proteins such as microtubule-associated protein 1 light chain 3 (LC3). Changes in intracellular localization of LC3 staining from the diffuse intracytoplasmic form (LC3-I) to the autophagosome-associated form linked to phosphatidylethanolamine (LC3-II) appeared as localized distinct puncta around the nucleus (41,42). A specific anti-LC3-II antibody (1:200; Abcam) was used to detect autophagic vacuoles.…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…633 Another possibility is immunohistochemical staining, an important procedure that may be applicable to human studies as well, considering the role of autophagy in neurodegeneration, myopathies and cardiac disease where samples may be limited to biopsy/autopsy tissue. Immunodetection of LC3 as definite puncta is possible in paraffin-embedded tissue sections and fresh frozen tissue, by either immunohistochemistry or immunofluorescence; 140,[634][635][636][637] however, this methodology has not received extensive evaluation, and does not lend itself well to dynamic assays. Other autophagic substrates can be evaluated via immunohistochemistry and include SQSTM1, NBR1, ubiquitinated inclusions and protein aggregates.…”