Abstract
Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM–HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM–HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM–HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.
Highlights
Human epidermal growth factor receptor 2 (HER2) is a member of receptor tyrosine kinase family comprised of EGFR (HER1), HER3, and HER4
For the last three decades, HER2 has been studied in great detail by clinicians and biologists, as HER2 gene amplification and protein overexpression have been associated with aggressive metastatic breast and ovarian cancers, as well as gastric and bladder cancers [1]
To the best of our knowledge, we report for the first time on the quantification of the binding of HER2 to TZM to monitor HER2–TZM engagement via Fluorescence lifetime imaging (FLI)-FRET microscopy (FLIM-FRET)
Summary
Human epidermal growth factor receptor 2 (HER2) is a member of receptor tyrosine kinase family comprised of EGFR (HER1), HER3, and HER4. Targeting HER2 with various therapeutic antibodies, e.g., trastuzumab (TZM). Pertuzumab, as well as antibody–drug conjugates (T-DM1) has led to a dramatic improvement in survival outcomes for HER+ cancer patients [2,3,4]. A significant number of eligible patients do not benefit from this therapy due to primary or acquired drug resistance. The molecular mechanisms of trastuzumab action and resistance are still not completely understood [5,6,7]. One of the possible reasons is insufficient target exposure and, as a result, reduced target engagement and poor drug efficacy. The ability to directly quantify antibody-HER2 engagement in the tumors may provide significant insights into whether drug therapy resistance is due to insufficient drug delivery in tumors
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