Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

Abstract

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. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. 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) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (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. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

Development of a carbon dot and methylene blue NIR-emitting FLIM-FRET pair in niosomes for controlled ROS generation

“…The versatile FLIM technique has been used several times earlier to locate biologically important lipid rafts in model membranes, 40,41 to study the interaction of biologically essential dyes in lipid vesicles, 42,43 in the preparation of fluorescent polymeric thermometers, 44 in monitoring fibril disintegration of insulin in the presence of cholic acid polymer 45 and also for protein sensing. 46,47 However, studies on CDs by FLIM is very limited. In one occasion, distribution of the CDs in cells was analysed by two-photon FLIM.…”

“…In this regard, we have utilized FLIM-FRET to obtain an amplified view with real heterogeneity. This method has been utilized earlier several times to detect nuclear protein adducts in plant cells, 52 for in vivo and in vitro tracking of receptor–ligand complexes in tumour cells, 47 to track protein localization in living cells and so on. 46 Here, we have used the lifetime changes of the donor YCDs in the presence of MB in niosomal dispersion to clarify the FRET process by FLIM.…”

The Opportunities and Use of Imaging to Measure Target Engagement

“…45 MFLI FRET provides a direct measurement of systemic delivery, target availability, and intracellular drug delivery in preclinical studies. 46 MFLI has been used to measure FRET longitudinally in intact and live animals. MFLI FRET is well suited for guiding the development of targeted drug therapy in heterogeneous tumors in intact, live small animals.…”