Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer

Abstract

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

[18F]DCP, First Generation PET Radiotracer for Diagnosis of Radiation Resistant Head and Neck Cancer

“…The mice were fasted for 6 hours prior to each set of optical experiments to maintain minimal variance in metabolic demand among animals (24). All measured mice received a tailvein injection of TMRE (100 µL of 75 µM) first and then a tail-vein injection 2-NBDG (100 µL of 6 mM 2-NBDG) with 20 minute delay as reported previously (25,64). The 2-NBDG uptake at 60 minutes post 2-NBDG injection and TMRE uptake at 80 minutes post TMRE injection were used to report final 2-NBDG and TMRE uptake.…”

“…Optical measurements on animals were performed by placing the fiber probe gently on the tumors and fixing the measurement on the center of the tumor mass (25). The measured spot size was 2.0-mm in diameter which was determined by the probe size as reported previously (25,64). Baseline diffuse reflectance and fluorescence spectra were measured from the tumor sites prior to any probe injection.…”

Optical Imaging of Glucose Uptake and Mitochondrial Membrane Potential to Characterize Her2 Breast Tumor Metabolic Phenotypes

“…The fluorescent indicators, 2-NBDG and TMRE, were chosen for our imaging due to their translatability to in vivo experiments our group have previously published (23,26,39,40). 2-NBDG Duke University Small Molecule Facility) is a fluorescent glucose analog that enters the cell via glucose transporters as a function of glucose uptake and causes an increase in fluorescence intensity (25,41).…”

“…Relying on endogenous fluorescence alone allows for imaging of flavin adenine dinucleotide (FAD) and reduced NADH to provide insight in the reduction-oxidation state in the electron transport chain with increased NADH fluorescence suggesting increased glycolysis and increased FAD fluorescence reflecting greater reliance on oxidative phosphorylation; however, they do not directly report on glucose uptake nor mitochondrial activity (22). Previ-ously, our group has developed a novel technique to image glucose uptake and mitochondrial membrane potential (mitochondrial metabolism) using the fluorescent probes, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), respectively (23). Our group (24)(25)(26)(27) and others (28)(29)(30) have extensively validated 2-NBDG imaging in cells (25), window chambers (24,26), and flank tumor models (27).…”

“…TMRE has been extensively used to study oxidative phosphorylation in vitro (31). Recently, we performed foundational studies to translate this indicator to quantitatively image oxidative phosphorylation in vivo (23,32). This platform could be valuable in informing the evolution of disease and can inform which stages of the cancer cascade could be most susceptible to metabolically targeted therapy.…”