Luciferases are widely used to monitor various biological processes. Here, we describe the naturally secreted Gaussia luciferase as a highly sensitive reporter for quantitative assessment of cells in vivo by measuring its levels in blood. The Gluc blood assay complements in vivo bioluminescence imaging which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in experimental disease and therapy models.Luciferase-mediated bioluminescence imaging has served as a reporting tool for monitoring various biological processes in vitro and in vivo in different fields 3 , including immunology 4 oncology 5 , virology 6 , and neuroscience 7 . After systemic substrate injection, a charge coupled device (CCD) camera can be used to localize the luciferase photon signals in vivo. Recently, we have characterized a naturally secreted luciferase from the marine copepod Gaussia princeps (Gaussia luciferase, Gluc) and found it to be over 2000-fold more sensitive than firefly and Renilla reniformis luciferases and 20,000-fold more sensitive than the secreted alkaline phosphatase (SEAP) when expressed in mammalian cells 8,9 . Gluc expression levels can be easily quantified in cell-free, conditioned medium by adding its substrate coelenterazine and measuring emitted photons using a luminometer. Since Gluc is secreted from mammalian cells in culture 9 , we hypothesized that it might also be secreted into the blood of animals harboring cells expressing this reporter.In order to assess the potential of Gluc as a reporter to monitor biological processes by measuring its level in the blood of small animals, we transduced Gli36 human glioma cells with a lentivirus vector encoding Gluc (Gli36-Gluc) and implanted them in different numbers into the flanks of nude mice. We visualized the tumors 3 days post-implantation by in vivo bioluminescence imaging after intravenous (i.v.) injection of the Gluc substrate, coelenterazine (4 mg/kg body weight) and acquiring photon counts using a CCD camera (Fig. 1a). At the same time, we withdrew 5 μl blood samples from these mice and directly aliquoted them into tubes containing EDTA (1 μl 20 mM), after which we measured the Gluc activity by adding coelenterazine (100 μM) and acquiring photon counts using a luminometer (Supplementary Methods online). The Gluc activity in the blood was linear with respect to cell number in a NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript range covering over 5 orders of magnitude, and correlated well with values obtained using the CCD camera (Fig. 1b). Further, Gluc activity could also be detected in the urine, albeit to a lesser extent than in the blood, which indicates it is cleared by the kidneys (Fig. 1b). In addition, there was no detrimental effect of EDTA on Gluc activity measured in blood ( Supplementary Fig. 1a online), and no significant differences were detected between the Gluc activity measured in serum or whole blood samples, showing that hemoglobin, which can int...

Normal differentiated cells rely primarily on mitochondrial oxidative phosphorylation to produce adenosine triphosphate (ATP) to maintain their viability and functions by using three major bioenergetic fuels: glucose, glutamine and fatty acids. Many cancer cells, however, rely on aerobic glycolysis for their growth and survival, and recent studies indicate that some cancer cells depend on glutamine as well. This altered metabolism in cancers occurs through oncogene activation or loss of tumor suppressor genes in multiple signaling pathways, including the phosphoinositide 3-kinase and Myc pathways. Relatively little is known, however, about the role of fatty acids as a bioenergetic fuel in growth and survival of cancer cells. Here, we report that human glioblastoma SF188 cells oxidize fatty acids and that inhibition of fatty acid β-oxidation by etomoxir, a carnitine palmitoyltransferase 1 inhibitor, markedly reduces cellular ATP levels and viability. We also found that inhibition of fatty acid oxidation controls the NADPH level. In the presence of reactive oxygen species scavenger tiron, however, ATP depletion is prevented without restoring fatty acid oxidation. This suggests that oxidative stress may lead to bioenergetic failure and cell death. Our work provides evidence that mitochondrial fatty acid oxidation may provide NADPH for defense against oxidative stress and prevent ATP loss and cell death.

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-13C]-glucose, [U-13C,15N]-glutamine, and [1,2- 13C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α–ketoglutarate through to fumarate. [1,2- 13C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.

Gaussia luciferase (Gluc) is a sensitive reporter for studying different biological processes such as gene expression, promoter activity, protein-protein interaction, signal transduction as well as tumor cell growth and response to therapy. Since Gluc is naturally secreted, the kinetics of these processes can be monitored in real-time by measuring an aliquot of conditioned medium in culture or a few microliters of blood in vivo at different time points. Gluc catalyzes light emission with a short halflife which is unfavorable for certain applications. We isolated a Gluc mutant that catalyzes enhanced light stability in the presence of a detergent, in combination with high sensitivity, making it an attractive luciferase for high-throughput functional screening applications.

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