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

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.

Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

“…The fluorescent lifetime decay graph ( Figure 3 B) and frequency graph ( Figure 3 C) demonstrate a significant reduction of donor lifetime in the presence of acceptor (A:D = 2:1), indicating FRET events due to HER2–TZM binding. As expected for FRET measurements of receptor–ligand interactions, e.g., TfR–Tf complexes [ 17 , 19 , 24 ], both HER2–TZM FRET donor fraction (FD%) and FRET efficiency ( E ) display rising trendlines when plotted against increasing A:D ratios ( Figure 3 D,E). Interestingly, the average FD% values are similar for both TfR–Tf and HER2–TZM complexes in cancer cells ( Supplementary Figure S3 ) [ 17 , 19 , 24 ].…”

“…As expected for FRET measurements of receptor–ligand interactions, e.g., TfR–Tf complexes [ 17 , 19 , 24 ], both HER2–TZM FRET donor fraction (FD%) and FRET efficiency ( E ) display rising trendlines when plotted against increasing A:D ratios ( Figure 3 D,E). Interestingly, the average FD% values are similar for both TfR–Tf and HER2–TZM complexes in cancer cells ( Supplementary Figure S3 ) [ 17 , 19 , 24 ]. To exclude molecular crowding effects, we have previously demonstrated that FLIM-FRET behaves independently from increasing acceptor concentration [ 16 , 31 ].…”

“…Furthermore, the FLI-based optical approach is amenable to imaging multiple biomarkers simultaneously, paving the way to not only quantifying drug target engagement but also drug response [ 23 ]. Recently, we reported on a new protocol to simultaneously image target engagement and glucose metabolism in an undisturbed tumor environment, which allows for the in vivo monitoring of ligand intracellular tumor uptake in combination with changes in tumor metabolic status in real time [ 24 ].…”

Luminescence lifetime imaging of three-dimensional biological objects

“…For example, FMT has been used in conjunction with structured light illumination to achieve quantitative 3D FRET tomographic non-invasive in vivo macroscopic FLI (MFLI) imaging of subcutaneous tumor xenografts to measure drug delivery (Rudkouskaya et al, 2018(Rudkouskaya et al, , 2020b. Subsequently, in vivo multiplexed MFLI-FRET imaging of receptor-ligand target engagement and glucose metabolism in tumor xenografts in live intact animals has been demonstrated (Rudkouskaya et al, 2020a). However, tomographic image reconstructions are computationally expensive and require expertise in light propagation modeling, limiting their use in biomedical applications (Leblond et al, 2010).…”

Deep Learning in Biomedical Optics

“…Fluorescence imaging has become a central tool in biomedical studies with high sensitivity to observe endogenous molecules [70,71] and monitor important biomarkers [72]. Increasingly, fluorescence imaging is not limited to intensity‐based techniques but can extract additional information by measuring fluorophore lifetimes [73–75].…”