KEYWORDS: Fluorescence resonance energy transfer, Antibodies, Fluorescence, Biological imaging, Tumors, Near infrared, Fluorescence imaging, In vivo imaging, Dyes, Target detection
Herein, we will use Macroscopy Fluorescence Lifetime Forster Resonance Energy Transfer (MFLI FRET) to quantitatively image in vivo and non-invasively the efficacy of receptor-targeted drug delivery systems. We have established an antibody-to-target imaging pipeline to develop novel near-infrared (NIR)-labeled probes to interrogate and quantify the dynamics of drug-receptor binding in HER2 breast cancer tumor xenografts.
KEYWORDS: Tumors, Fluorescence resonance energy transfer, In vivo imaging, Fluorescence, Near infrared, Animal model studies, Optical imaging, Collagen, Tissues, Resonance energy transfer
Macroscopic fluorescence lifetime Forster resonance energy transfer (MFLI FRET) imaging presents an analytical tool to non-invasively quantify drug-receptor engagement in tumors and other organs in preclinical studies. Near infrared (NIR) fluorescence lifetime FRET acts as a direct reporter of drug-target engagement via measuring the fraction of labeled-donor antibody drugs undergoing binding to their respective target receptor using in vitro microscopy or in vivo macroscopy. We show that NIR MFLI FRET measurements correlates with drug-receptor binding in tumor cells, but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Our study demonstrates that MFLI FRET is a powerful non-invasive imaging approach to study the impact of tumor microenvironment on drug delivery and drug-target engagement in intact live tumor xenografts.
KEYWORDS: Tumors, Fluorescence resonance energy transfer, Resonance energy transfer, Fluorescence imaging, Fluorescence, Breast, Antibodies, Fluorophores, In vivo imaging, Dyes
Emergent breast tumor resistance and tumor microenvironment (TME) heterogeneity can lead to decreased drug delivery efficacy, resulting in therapeutic failure. Preclinical molecular imaging is a crucial tool in the advancement of targeted therapeutics for supporting the development of new drugs but also to elucidate factors hampering optimal drug delivery. However, noninvasive tumor imaging modalities that can quantify drug-target engagement, which is critical for therapeutic actuation, are still lacking. We have demonstrated the utility of macroscopic fluorescence lifetime Forster’s Resonance Energy Transfer (MFLI FRET)-based optical imaging to measure labeled trastuzumab (TZM)-human epidermal growth factor receptor (HER2) binding in human HER2+ cell lines and breast tumor xenograft mice models. We have established a clinically relevant TZM antibody drug containing the Meditope (MDT) peptide conjugated to near-infrared (NIR) dyelabeled FRET pairs, that retain full HER2 binding capability. Herein, we demonstrate FRET measurements using MFLI in vivo imaging platform to measure the ability of MDT-TZM to bind HER2 in living breast tumor xenografts. HER2+ AU565 breast tumor xenografts bearing nude mice were injected retro-orbitally with TZM (NHS-conjugated) or MDTTZM labeled with AlexaFluor700 (donor) and AlexaFluor750 (acceptor) and MFLIFRET imaging was performed 24 h and 48 h post-injection. Preliminary data suggest that MDT-TZM shows higher uniform and consistent FRET signal compared to TZM, suggesting increased efficacy of TZM-MDT-HER2 binding. Also staggered injections of donor and acceptor MDT-TZM may be optimal for quantifying MDT-TZM using MFLIFRET compared to single injections.
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.
KEYWORDS: Tumors, Fluorescence resonance energy transfer, Near infrared, Fluorescence lifetime imaging, Signal detection, Resonance energy transfer, Microscopy, Imaging systems
There is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies in oncology. We have established Macroscopic Fluorescence Lifetime Imaging (MFLI) associated with Forster Resonance Energy Transfer (FRET) to report quantitatively on antibody-target engagement in live intact animals at the organ level. Here, we use FLI FRET imaging to quantify the binding of near infrared labeled antibody drugs to breast cancer cells or tumor xenografts. In combination with antibody engineering, this approach provides a robust analytical tool to optimize the binding and uptake of antibody-target binding during tumor progression and treatment.
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