Flourescence Resonance Energy Transfer as a Measure of HER-2/HER-3 Dimerization in Paraffin-Fixed, Antibody-Stained Breast Cancer Tissue
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Expression of the human epidermal growth factor receptor 2 (HER-2) gene is dysregulated in 20-30% of breast cancers and has been established as an independent prognostic factor and a predictive indicator of treatment efficacy. However, HER-2- mediated signaling is contingent upon dimerization with HER-3, a member of the same tyrosine kinase receptor family. Thus, measurement of the physiologically functional HER-2/HER-3 unit, rather than HER-2 alone, is proposed as a more accurate prognostic and diagnostic tool. This study attempts to measure HER-2/HER-3 dimerization in paraffin-fixed, antibody-stained breast cancer tissue using fluorescence resonance energy transfer (FRET) technology, which can quantify protein-protein interactions as determined by intermolecular distances. Ten randomly selected breast tumor tissues were stained for the HER-2 and HER-3 proteins with primary antibodies conjugated to fluorophore-tagged secondary antibodies. Images of the tissues were taken with a laser scanning microscope to quantify dimerization, represented as a measure of fluorescence due to energy transfer between the fluorophores. Our data suggests measuring FRET between HER-2 and HER-3 is not feasible in fixed tissue and lends support to the development of an in vitro model of HER-2/HER-3 dimerization for more accurate quantification.