Abstract
Olink Bioscience has developed the Duolink® assay, which allows precise detection and quantification of proteins, protein interactions and modifications in fixed cells and tissue samples in their correct cellular context at physiologically relevant expression levels. This tool may ultimately be used in conjunction with new-generation anti-cancer therapies for inhibiting specific receptor dimerizations.
Main
The importance of the human epidermal growth factor receptor (HER) family in the development and progression of various cancers is widely recognized. Standard immunohistochemistry yields acceptable semi-quantitative data for total protein levels; however, it cannot be used to analyze receptor dimerization patterns. To allow detailed studies of protein-protein interactions, we have developed Duolink, a product based on in situ PLA®. Duolink detects proximal proteins in their normal context at normal expression levels and also allows rare events to be detected by generating cell-to-cell statistics1. Here we give an example of how Duolink can be used to study epidermal growth factor (EGF) receptor (EGFR) and HER2 receptor dimerization and activation patterns in cells and tissue samples. Depending on the interaction partner for a specific HER receptor, different signaling cascades are activated. Measuring HER-family interaction patterns may have great potential as a companion diagnostic for the new-generation anti-cancer therapies aimed at inhibiting specific receptor dimerizations2.
The Duolink reagents and in situ PLA technology
The Duolink kit series allows the user to combine any pair of immunofluorescence- or immunohistochemistry-validated antibodies. Duolink readout is performed with either a fluorescent label (for fluorescence microscopy) or horseradish peroxidase (HRP; for bright-field detection). The resulting distinct spots are derived from single-molecule protein-interaction events (Fig. 1).
Studying HER-family receptor dimerization and activation in cells
At least 11 different ligands are known that regulate HER-family receptor activation and dimerization2. Here we used EGF, which exclusively binds and phosphorylates EGFR, to validate EGFR-HER2 dimerization and activation. Two different cell lines, SKBR3 (known to express EGFR and high levels of HER2) and MDA-MB-468 (known to express EGFR and very low levels of HER2), were stimulated with the ligand EGF. Both cell lines responded to EGF stimulation by phosphorylation of EGFR (Fig. 2), which was measured using an anti-EGFR antibody (Y-1068). However, only the SKBR3 cell line gave a detectable signal after EGF stimulation when the interaction between phosphorylated EGFR (pEGFR) and HER2 was measured. This was expected, given that the MDA-MB-468 cell line has very low levels of HER2 expression. To further confirm that EGFR-HER2 interactions took place, phosphorylated HER2 (pHER2) was measured using an anti-HER2 (Y1221/1222) antibody. EGF does not bind to HER2; therefore, HER2 can be activated after EGF stimulation only via a complex with EGF-stimulated EGFR. A strong phosphorylation of HER2 was observed. The use of an antibody targeting pEGFR instead of total EGFR ensured that it was an activated EGFR-HER2 complex that was measured and not an inactive, preformed complex that does not elicit downstream signaling. The existence of inactive, preformed complexes has been suggested in the literature3,4.
Studying tyrosine kinase receptor dimerization and activation in tissue
An advantage of Duolink compared to systems based on tagged proteins is that an assay validated on cells can be transferred and applied directly to tissue samples. In Figure 3, two different tissue samples shown to contain high levels of pEGFR were assayed for pEGFR-HER2 complexes using the same assay as shown on cells in Figure 2. Only one of the samples exhibited high levels in the pEGFR-HER2 assay. The presence of a complex between phosphorylated EGFR and HER2 would lead to phosphorylation of the HER2 receptor. A Duolink assay measuring pHER2 accordingly showed high levels of pHER2 in the same tissue areas showing high levels of pEGFR-HER2 complexes (Fig. 3e). Another example of how Duolink can be used to study HER-family interactions was recently published for the HER2-PTK6 complex detected in FFPE material5. The result indicates prognostic relevance of the HER2-PTK6 complex, showing the importance of investigating protein-interaction events for diagnostic use.
The concept described here is of course applicable to any receptor in the HER family. Care need only be taken that the two antibodies accept compatible antigen-retrieval conditions and are still able to bind their target when the target protein is interacting with its partner.
Conclusions
The Duolink assay offers a unique opportunity to decipher HER-family receptor interactions. It holds great promise for further fine-tuning companion diagnostics for HER family receptor–targeted therapies and as a means of measuring the effects of new drugs targeting specific interactions. In situ PLA using Duolink reagents is a straightforward process for reporting protein interactions and homodimerization events with very high specificity in their natural context at physiological expression levels.
References
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Gullberg, M., Huang, S. & Andersson, AC. Analyzing human epidermal growth factor receptor family dimerization and activation using Duolink®. Nat Methods 7, 10–11 (2010). https://doi.org/10.1038/nmeth.f.318
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DOI: https://doi.org/10.1038/nmeth.f.318