Near infrared fluorescence lifetime FRET microscopy to evaluate antibody drug binding in various HER2 positive cancer cell lines

Catherine Sherry; Amit Verma; Jason Smith; Vikas Pandey; Tynan Young; John Williams; Xavier Michalet; Xavier Intes; Margarida Barroso

doi:10.1117/12.2654748

25 April 2023 Near infrared fluorescence lifetime FRET microscopy to evaluate antibody drug binding in various HER2 positive cancer cell lines

Catherine Sherry, Amit Verma, Jason Smith, Vikas Pandey, Tynan Young, John Williams, Xavier Michalet, Xavier Intes, Margarida Barroso

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Proceedings Volume 12384, Multiphoton Microscopy in the Biomedical Sciences XXIII; 123840P (2023) https://doi.org/10.1117/12.2654748
Event: SPIE BiOS, 2023, San Francisco, California, United States

Abstract

We aim to use the resolving power of near-infrared (NIR) fluorescence lifetime microscopy (FLIM) to provide information on the fluorescence decay behavior of NIR FRET donor probes, both in solution and in vitro, and assess their impact on in vivo macroscopic FLI FRET (MFLI FRET) tumor imaging. Utilizing HER2 mAbs, i.e., trastuzumab (TZM), labeled with AlexaFluor 700 (AF700), and HER2 positive cancer cell lines (AU565 and SKOV-3), we have documented significant impacts of IRF extraction methods and probe labeling schemes on FLIM analysis. Additionally, we have noted marked variation in the intracellular distribution of the HER2-TZM binding complexes, as well as in average endosomal lifetime measurements between cell lines. Herein, we discuss optimal methods for IRF extraction and generating NIR probes, as well as results from the newly optimized NIR FLIM FRET assay demonstrating variations in the average intracellular lifetime of TZM-AF700. Because fluorescence lifetime is impacted by environmental factors, such as pH, refraction, viscosity, and proximity to other molecules, these variations imply differences in the way TZM interacts with the endosomal microenvironment of these cell lines. We hypothesize that different HER2 positive cancer types exhibit variations in endosomal trafficking of the HER2-drug complex that play a key role in primary/acquired resistance to TZM.

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Catherine Sherry, Amit Verma, Jason Smith, Vikas Pandey, Tynan Young, John Williams, Xavier Michalet, Xavier Intes, and Margarida Barroso "Near infrared fluorescence lifetime FRET microscopy to evaluate antibody drug binding in various HER2 positive cancer cell lines", Proc. SPIE 12384, Multiphoton Microscopy in the Biomedical Sciences XXIII, 123840P (25 April 2023); https://doi.org/10.1117/12.2654748

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KEYWORDS

Fluorescence resonance energy transfer

Fluorescence

Near infrared

Fluorescence lifetime imaging

In vitro testing

Dyes

Fluorophores

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