coelenterazine and Breast-Neoplasms

The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; Female; Fluorescence Resonance Energy Transfer; Humans; Imidazoles; Luciferases; Luminescent Agents; Luminescent Measurements; Mice; Mice, Inbred NOD; Mice, SCID; Optical Imaging; Pyrazines; Spectroscopy, Near-Infrared; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Amino Acid Sequence; Animals; Binding Sites; Breast Neoplasms; Chlorocebus aethiops; COS Cells; Female; Fluorescence Resonance Energy Transfer; HeLa Cells; Heterografts; Humans; Imidazoles; Liver Neoplasms; Luciferases; Luminescent Agents; Luminescent Measurements; Luminescent Proteins; Lung Neoplasms; Mice; Models, Molecular; Molecular Imaging; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Pyrazines; Receptors, Estrogen; Recombinant Fusion Proteins; Red Fluorescent Protein; Single-Cell Analysis

The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.

Topics: Biological Transport; Breast Neoplasms; Cell Line, Tumor; Extracellular Vesicles; Female; Genes, Reporter; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Imidazoles; Luciferases, Renilla; Luminescent Measurements; Nanoparticles; Pyrazines; Recombinant Fusion Proteins; Sensitivity and Specificity; Subcellular Fractions; Tetraspanin 29; Tetraspanin 30

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β 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 a 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.

Topics: Animals; Breast Neoplasms; Cell Line; Disease Progression; Female; Heterografts; Imidazoles; Luciferases; Luciferases, Firefly; Luminescent Measurements; Mice; Molecular Imaging; Neoplasm Transplantation; Pyrazines; Signal Transduction; Substrate Specificity; Transfection; Transforming Growth Factor beta

Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine[#x02013]chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase (GL), an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl concentration. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds that specifically target CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.

Topics: Animals; Biological Assay; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chemokine CXCL12; Copepoda; Drug Discovery; Female; Genetic Vectors; Green Fluorescent Proteins; Humans; Imidazoles; Kidney; Lentivirus; Luciferases; Luminescent Measurements; Luminescent Proteins; Promoter Regions, Genetic; Protein Binding; Pyrazines; Receptors, CXCR; Receptors, CXCR4; Recombinant Fusion Proteins; Signal Transduction; Substrate Specificity; Transduction, Genetic