Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

Benjamin R. Waterhouse; Merel Gijsen; Paul R. Barber; Iain D.C. Tullis; Borivoj Vojnovic; Anthony Kong

doi:10.18632/oncotarget.313

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Methodological Reports:

Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

Benjamin R. Waterhouse, Merel Gijsen, Paul R. Barber, Iain D.C. Tullis, Borivoj Vojnovic and Anthony Kong _

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Oncotarget. 2011; 2:728-736. https://doi.org/10.18632/oncotarget.313

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Abstract

Benjamin R. Waterhouse^1*, Merel Gijsen^1*, Paul R. Barber², Iain D.C. Tullis², Borivoj Vojnovic² and Anthony Kong^1*

¹Human Epidermal Growth Factor Receptor Group; Cancer Research UK Molecular Oncology Laboratories, The Weatherall Institute of Molecular Medicine (WIMM), University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.

²Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Churchill Hospital, Oxford, OX3 7DQ, UK

^* Indicates equal contribution

Received: August 26, 2011; Accepted: September 6, 2011; Published: September 8, 2011;

Keywords: HER (ErbB) receptors, EGFR, HER2 (ErbB2), dimerization, FRET, FLIM

Correspondence:

Anthony Kong, email:

Abstract

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.

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Methodological Reports:

Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

Abstract