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Theranostics 2020; 10(22):10309-10325. doi:10.7150/thno.45825 This issue Cite

Research Paper

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

Alena Rudkouskaya¹, Nattawut Sinsuebphon^2#, Marien Ochoa², Sez-Jade Chen², Joseph E. Mazurkiewicz³, Xavier Intes², Margarida Barroso^1✉

1. Department of Cellular and Molecular Physiology, Albany Medical College, Albany, NY 12208, USA.
2. Center for Modeling, Simulation, and Imaging in Medicine, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
3. Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, NY 12208, USA.
#Current address: Assistive Technology and Medical Devices Research Center, National Science and Technology Development Agency (NSTDA), Khlong Luang, Pathum Thani, 12120, Thailand.

✉ Corresponding author: Margarida Barroso, E-maiL: barrosmedu;

Abstract

Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes.

Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results.

Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery.

Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

Keywords: target engagement, metabolism, breast cancer, lifetime imaging, FRET

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