Monitoring receptor heterodimerization along intracellular trafficking pathways using anti-HER2 therapeutic antibodies

Abstract

Overexpression of Human EGF Receptor 2 (HER2) in cancer is a marker of aggressive metastatic disease and poor prognosis. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used in the clinic over the last decades. However, a large fraction of eligible patients display resistance to this therapy. This calls for a deeper investigation of HER2 interaction with other members of HER tyrosine kinase receptors and modulation of their endocytic trafficking upon TZM treatment. Forster resonance energy transfer Fluorescence lifetime microscopy (FLIM- FRET) offers a robust approach to monitor HER2 homo and heterodimerization via the reduction of donor fluorophore lifetime. The objective of this study was to assess the dynamics of HER receptor homo and heterodimerization behavior via FLIMFRET by using near-infrared (NIR) FRET pair labeled anti-HER2 and anti-EGFR therapeutic antibodies in HER2- overexpressing breast cancer cells. In addition, we tested our new deep learning platform DL4FLIM adapted for automated analysis of all datasets. Herein, we report a first attempt to quantify NIR FRET pair labeled cetuximab (CTM, as a donor) and TZM (as an acceptor) binding to their receptors EGFR and HER2 respectively in AU565 cells. As a control, we also performed and human isotype IgG FLIM -FRET and found it completely non-specific. Our data demonstrate both the occurrence of FRET between NIR-labeled probes CTM and TZM as well as between CTM-CTM bound to their respective receptors. This proof-of -principal study demonstrated feasibility of monitoring HER2 hetero receptor FRET FLIM to better understand mechanism of TZM resistance.