Quantification of anti-HER2 drug uptake into human breast cancer cells using fluorescence lifetime FRET imaging

Alena Rudkouskaya; Jason Smith; Marien Ochoa; Xavier Intes; Margarida Barroso

doi:10.1117/12.2544429

19 February 2020 Quantification of anti-HER2 drug uptake into human breast cancer cells using fluorescence lifetime FRET imaging

Alena Rudkouskaya, Jason Smith, Marien Ochoa, Xavier Intes, Margarida Barroso

Author Affiliations +

Proceedings Volume 11219, Visualizing and Quantifying Drug Distribution in Tissue IV; 112190C (2020) https://doi.org/10.1117/12.2544429
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

Human EGF receptor 2 (HER2) is an important oncogene and marker of aggressive metastatic cancer, found in up to 20% of oncologic patients. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used over the last two decades. However, both primary and acquired TZM resistance calls for the deeper investigation on TZMHER2 binding, internalization and trafficking/degradation in cancer cells in vitro and in vivo. Fluorescence lifetime FRET imaging (FLIM FRET) offers a unique approach to monitor TZM-HER2 binding followed by their uptake into target cells via the reduction of donor fluorophore lifetime. In this study, we characterized for the first time TZM-AF700 uptake and its relation to HER2 expression in AU565 human breast cancer cell line using confocal microscopy. Further, we have quantified the dimerization of HER2 via NIR FLIM FRET in vitro microscopy. Extensive analysis confirmed high specificity and efficiency of TZM FRET signal. Interestingly, we observed a significant heterogeneity of FRET within the cells: the highest TZM FRET levels occurred at the plasma membrane, whereas less if any donor lifetime reduction was registered in the perinuclear endosomes. These results suggest that HER2 dimers undergo dissociation or degradation upon TZM binding and trafficking. Overall, this study provides a good foundation for in vivo TZM FRET imaging of target engagement in preclinical studies.

Conference Presentation

Citation Download Citation

Alena Rudkouskaya, Jason Smith, Marien Ochoa, Xavier Intes, and Margarida Barroso "Quantification of anti-HER2 drug uptake into human breast cancer cells using fluorescence lifetime FRET imaging", Proc. SPIE 11219, Visualizing and Quantifying Drug Distribution in Tissue IV, 112190C (19 February 2020); https://doi.org/10.1117/12.2544429

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