We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF- signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. biology, use of a single luciferase means that only one molecular event or cell population can be analyzed. Firefly and other beetle luciferases also are relatively large enzymes ( 60 kDa) that require ATP as a co-factor, creating potential steric problems when fused to other proteins and restricting applications to intracellular events. To overcome limitations of beetle luciferases and expand the range of intracellular and extracellular events that can be imaged, there is an ongoing need for smaller, Befetupitant ATP-independent luciferases for use in fusion proteins and analyses of extracellular molecules. Ideally, new luciferase reporter enzymes would have substrates and emission spectra distinct from beetle luciferases to enable monitoring of two distinct events, similar to fluorescence techniques. luciferase (GL) is used as an alternative or complement to beetle luciferases for cell-based and animal imaging studies. GL has a molecular weight ( 20 kDa) smaller than fluorescent proteins, so fusing this enzyme to a protein of interest is less likely to perturb functions of a target protein as compared with beetle luciferases. As Rabbit Polyclonal to OR8J3 an ATP-independent, secreted enzyme, GL provides a marker for extracellular signaling, protein secretion, and tumor growth (6C8). GL also uses a different substrate, coelenterazine, than the pair of firefly luciferase and luciferin, enabling imaging of two different molecular or cellular events. However, GL has notable disadvantages that limit its use for animal imaging studies. Coelenterazine oxidizes in serum, producing high levels of background luminescence that decrease detection of imaging signals in cultured cells and living animals. GL also exhibits flash kinetics, such that the signal drops by approximately 70% within 1 minute (6). Mutants of GL with more sustained, glow-like kinetics have been described recently, although these mutants require special assay conditions incompatible with intact cells or exhibit dramatic (100X) losses in bioluminescence (9, 10). Coelenterazine must be injected systemically via intravenous or intracardiac injection for animal imaging, which substantially restricts animal throughput. Befetupitant Finally, GL emits blue light with peak emission at 480 nm, which limits penetration through tissues for imaging. As an alternative to GL as a small, secreted, ATP-independent luciferase, we investigated a recently reported luciferase from the deep sea shrimp engineered for enhanced protein stability (NanoLuc, NL) and its optimally designed substrate, furimazine (11). NL is a 19 kDa, ATP-independent enzyme with some properties suggesting it will be advantageous for molecular imaging studies as compared with GL. In particular, NL has sustained glow-type luminescence, providing a broader time window for imaging. Furimazine, a derivative of coelenterazine optimized for NL, has greater stability and reduced background activity to enhance signal detection. In addition, by appending a secretion signal to NL, the enzyme is secreted efficiently into the extracellular space. In the current study, we show that bioluminescence imaging for NL can be accomplished in superficial tumors and internal organs of living mice with sensitivity at least comparable to GL. NL signal increases in proportion to tumor growth, providing an imaging metric for tumor burden. NL also can be detected readily in small samples of serum from living mice. Finally, we establish Befetupitant that NL can be combined with FL to image two distinct signaling events in a tumor, expanding the capabilities for molecular imaging in small animal.