Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumor EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labeling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within one-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.

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...

Application of bioluminescence imaging has grown tremendously in the past decade and has significantly contributed to the core conceptual advances in biomedical research. This technology provides valuable means for monitoring of different biological processes for immunology, oncology, virology and neuroscience. In this review, we will discuss current trends in bioluminescence and its application in different fields with emphasis on cancer research.