Quantification of HER2 dimerization in human breast cancer cells using fluorescence lifetime FRET microscopy and nanobody probes

Epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is associated with aggressive disease. HER2- targeted therapies such as trastuzumab (TZM) have improved outcomes, but drug resistance remains a significant barrier to effective treatment. Understanding HER2 receptor dimerization and trafficking is critical for improving therapeutic response. We used near-infrared (NIR) fluorescence lifetime microscopy (FLIM) FRET with anti-HER2 nanobodies to quantify relative changes in HER2 dimerization. Nanobodies are small, single-domain antibodies derived from the immune system of camelids. Nanobodies were conjugated to NIR donor or acceptor fluorophores and applied to live in HER2- overexpressing breast cancer cells. We leverage the differential sensitivity of the FRET fractional amplitude (A1) versus FRET efficiency (E) to distinguish receptor dimerization from probe-induced clustering. FLIM FRET imaging suggests that nanobody probes quantify native HER2 homodimerization through the amplitude of the short lifetime signal (A1). Antibody probes report on probe conformation and nanoscale packing through FRET efficiency (E), while showing no variation in A1. HER2 nanobody FLIM FRET provides a novel approach to investigate unperturbed HER2 dimerization and trafficking dynamics in cancer cells and inform mechanisms of therapeutic resistance.