Abstract B48: Optical toolbox for in vivo analysis of glucose uptake, vascular oxygenation, and mitochondrial membrane potential in breast cancer
Authors: Martinez Amy Frees , McCachren Samuel S. , Rajaram Narasimhan , Dewhirst Mark W. , Ramanujam Nimmi 
Published: May 2016

Abstract
The goal of this work is development of a fast and repeatable optical assay that uses a novel combination of fluorophores and endogenous contrast to report on the metabolic phenotype of cancers in vivo. The proportions of ATP generated by glycolysis and oxidative phosphorylation relate to inherent tumor behaviors that affect treatment response. The need for instruments measuring metabolic endpoints is demonstrated by the widespread use of Seahorse extracellular flux analyzers in cancer research. The assays provide valuable insight into two primary arms of cellular metabolism by reporting on extracellular acidification rate (ECAR) related to glycolysis and oxygen consumption rate (OCR) related to mitochondrial respiration [1]. The ratio OCR/ECAR obtained by Seahorse was used to compare a panel of breast cancer cell lines and show that basal subtypes were often highly glycolytic, which suggested better therapeutic outcome [2]. While the Seahorse is an excellent research tool, the technique requires special cell preparation and continual testing of cell media, and is therefore not applicable in vivo. Being able to measure both oxidative and glycolytic metabolism in vivo in a repeatable way would aid the identification of appropriate therapies based on tumor phenotype.
We developed an in vivo metabolic imaging strategy for use on a dorsal skin flap chamber model and are developing tools to analyze the same endpoints in solid tumors [3-5]. Vascular oxygen saturation (SO2) is calculated using endogenous absorption contrast from oxy- and deoxy- hemoglobins (520-620nm in 10nm increments). Glucose uptake is measured using a fluorescent D-glucose derivative (2-NBDG, Ex/Em 470/525nm). We have previously optimized 2-NBDG imaging [4], and have recently incorporated a fluorescent cationic dye (TMRE, Ex/Em 545/590nm) to report on mitochondrial membrane potential (MMP). Delivery-corrected 2-NBDG was measured 60 minutes post-injection (2-NBDG60/RD) and TMRE was measured 50 minutes post-injection (TMRE50) in dorsal windows containing metastatic (4T1) or non-metastatic (4T07, 67NR) sibling tumor lines.
When images were segmented into spatial maps and parsed by regional SO2, phenotypic differences between metastatic and nonmetastatic tumors emerged. To increase the range of SO2, some animals were subject to hypoxia. In 4T1 tumors, glucose uptake decreased significantly (p&lt;0.01 for 0&lt;SO2&lt;10 vs. 40&lt;SO2&lt;60) and MMP increased slightly as SO2 increased. However, in 4T07 and 67NR tumors there is minimal change in glucose uptake or MMP with changes in SO2.
Differences in global averages were also apparent. Glucose uptake was significantly increased in 4T1 relative to either 4T07 (p&lt;0.01) or nontumor tissue (p&lt;0.02). Interestingly, glucose uptake was comparable between 4T07 and nontumor. On the other hand, MMP was increased in 67NR (p&lt;0.02) and 4T1 (p&lt;0.01) relative to nontumor, suggesting generally increased oxidative metabolism in tumors. MMP was indistinguishable for 67NR and 4T1. Average SO2 was comparable across groups. Strikingly, the ratio of average glucose uptake/average MMP was 0.45 for metastatic and 0.12 for nonmetastatic tumors. These data indicate the importance of considering both the overall quantity of metabolic demand and the ratio of glycolytic and oxidative metabolism.
In conclusion, our data show a shift toward glycolysis in a metastatic relative to a nonmetastatic sibling breast cancer line, demonstrating the potential of our method to identify unique metabolic phenotypes in vivo. Our future efforts focus on transitioning the assay into an optical spectroscopy platform appropriate for long-term monitoring of tumor growth or therapy response.
[1] Seahorse Bioscience online
[2] Dennison, Clin Cancer Res 2013
[3] Rajaram, PLOS ONE 2013
[4] Frees, PLOS ONE 2014
[5] Rajaram, PLOS ONE 2015
Citation Format: Amy Frees Martinez, Samuel S. McCachren, III, Narasimhan Rajaram, Mark W. Dewhirst, Nimmi Ramanujam. Optical toolbox for in vivo analysis of glucose uptake, vascular oxygenation, and mitochondrial membrane potential in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B48.