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

In this study we investigate anti-HER2 therapeutic antibodies binding, internalization and trafficking in 2D and 3D AU565 breast cancer cells systems using fluorescence lifetime FRET microscopy. This enables the direct measurement of target engagement by sensing the reduction of donor fluorophore lifetime. In addition, it allows monitoring the dynamics of probe lifetime changes during endocytic trafficking, and even assess the heterodimerization pattern of HER receptors upon drug treatment by using different combinations of NIR labeled anti-HER1, -HER2 and -HER3 probes as donors and acceptors. Together, FRET FLIM provides a unique analytical tool to address challenges associated with anti-HER2 drug resistance.