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 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. Deeper investigation of TZM-HER2 binding, internalization and trafficking/degradation in cancer cells may provide some crucial answers. Forster resonance energy transfer Fluorescence lifetime microscopy (FRET-FLIM) offers a unique approach to monitor TZM-HER2 binding via the reduction of donor fluorophore lifetime. Thus, FRET-FLIM provides an efficient method to quantify ligand-receptor binding and internalization in cancer cells with the additional benefit of sensing intracellular microenvironment heterogeneity such as modulation of endocytic trafficking upon drug treatment. In this study, we report quantification of binding of NIR-labeled therapeutic antibodies FRET pairs to their respective receptors, e.g. HER2 and/or HER1, in AU565 breast cancer cells in various combinations, i.e. anti-HER1 cetuximab donor – cetuximab acceptor vs. cetuximab donor - TZM acceptor, to assess the dynamics of HER receptor homo and heterodimerization behavior. We also monitor FRET FLIM events along the intracellular trafficking of these anti-HER2 and anti-HER1 probes. Our data indicate that in the presence of TZM, HER1 as well as HER3 receptors localization is shifted towards plasma membrane. Furthermore, the distribution pattern of fluorescence lifetime during endocytic trafficking differs when comparing “homo” vs “hetero FRET” pairs. These results suggest that HER2 dimerization pattern can switch towards hetero- and oligomerization during TZM treatment which may result in modulation of oncogenic signaling. In parallel, Western blotting and immunofluorescent analysis of AU565 cells strongly indicate that 24h TZM treatment substantially upregulate HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Together, these data imply that in cultured breast cancer models, short-term TZM treatment may inadvertently increase oncogenic fitness probably as a result of adaptation to HER2 - TZM binding.