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.
Highlights
Deregulation of cellular energetics is a hallmark of cancer[1], and metabolic profiling of tumors allows researchers to investigate the mechanisms underlying cancer progression, metastasis, and resistance to therapies[2,3,4,5]
Some studies have found that dormant cells exhibit a relatively increased dependence on mitochondrial metabolism[17,18], confirming that the ability of tumor cells to shift their metabolism between glycolysis and oxidative phosphorylation is essential for survival in changing environments[19]
We have developed a novel strategy to image the spatiotemporal relationship between glucose uptake and mitochondrial metabolism in an intact tissue microenvironment using intravital microscopy
Summary
Deregulation of cellular energetics is a hallmark of cancer[1], and metabolic profiling of tumors allows researchers to investigate the mechanisms underlying cancer progression, metastasis, and resistance to therapies[2,3,4,5]. Tumors with increased capacity for both glycolysis and oxidative phosphorylation tend to be aggressive, with the ability to survive stressors such as cycling hypoxia or low nutrient availability. Some studies have found that dormant cells exhibit a relatively increased dependence on mitochondrial metabolism[17,18], confirming that the ability of tumor cells to shift their metabolism between glycolysis and oxidative phosphorylation is essential for survival in changing environments[19]. The Seahorse assay measures two metabolic endpoints: the extracellular acidification rate (ECAR), which reports indirectly on glycolysis, and oxygen consumption rate (OCR), which reports on oxidative phosphorylation These assays are useful in high-throughput experiments and can be used to compare the ratio of glycolytic to oxidative metabolism across a spectrum of cell types[32,33]
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