Triple bioluminescence imaging for in vivo monitoring of cellular processes

Abstract

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.Molecular Therapy - Nucleic Acids (2013) 2, e99; doi:10.1038/mtna.2013.25; published online 18 June 2013.

Figure 1

In vitro characterization of Vluc-catalyzed luminescence. (a) Vluc assay was performed on conditioned medium as well as cell lysates from 293T cells transduced with a lentivirus vector encoding Vluc to determine the fraction (in RLU) of Vluc being secreted (n = 3; *P = 0.003). (b) Vluc light emission kinetics was performed by adding the vargulin substrate to Vluc-containing media (n = 3). (c) Stability of Vluc enzyme was determined by assaying an aliquot of Vluc-containing cell-free conditioned medium, incubated at 37 °C, at different time points (n = 3). The slight increase observed between day 0 and 4 is not statistically significant (P = 0.176). (d) Vluc-based viability assay. 10⁵ U87 cells expressing Vluc were seeded in 12-well plates and an aliquot of conditioned medium was assayed for Vluc activity over time. For comparison, a commercially available Fluc-based viability assay was performed on the same cells from which Vluc-media was isolated (n = 3 per time point). RLU, relative light unit.

Figure 2

Triple in vivo bioluminescence imaging. (a) 2 × 10⁶ U87 glioma cells stably expressing Vluc were implanted subcutaneously in nude mice. Tumor-associated Vluc signal after iv or ip injection of 4 mg/kg vargulin is shown by a representative mouse from each injection route (n = 3). (b) U87 glioma cells stably expressing Gluc, Vluc or Fluc were injected subcutaneously in nude mice at different sites. Ten days later, sequential imaging of Fluc, Gluc, and Vluc was performed (1 day apart) after injection of D-luciferin, coelenterazine, and vargulin. A representative mouse from each imaging session is shown (n = 3). (c) A region of interest (ROI) was drawn around each tumor location and photons/second were calculated.

Figure 3

Monitoring of sTRAIL-mediated gene therapy with triple bioluminescence imaging. (a) Schematic diagram for the different AAV (left) and lentivirus (right) vector constructs used in this study. AAV vectors: CMV E, cytomegalovirus enhancer; CBA, chicken β-actin promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; pA, bGH poly A signal. Lentivirus vectors: Diagrams shown represent integrated provirus. CMV IE, cytomegalovirus immediate early promoter; cHS4 INS, chicken β globin hypersensitive site 4 insulator sequence; NF, nuclear factor-κB inducible promoter. (b–f) 10⁵ U87-Fluc/NF-Gluc glioma cells were implanted intracranially in nude mice. Nineteen days later, the mice brains were infused at the same tumor injection site with 10¹⁰ gc of either AAV-Vluc/GFP, AAV-Vluc/sTRAIL, or AAV-GFP alone. (b) Vluc bioluminescence imaging performed 10 days after vector injection confirming AAV-mediated gene delivery. (c) Two weeks post-vector injection, tumor volume was imaged by Fluc bioluminescence imaging. (d) Confirmation of NF-Gluc construct. U87-Fluc/NF-Gluc cells in culture were treated with or without tumor necrosis factor-α (TNF-α) (10 ng/ml). Forty-eight hours later, conditioned medium were collected and assayed for Gluc activity to detect levels of NF-κB induction (*P = 0.0001). (e,f) NF-Gluc imaging, a marker for TRAIL binding on glioma cells performed after iv injection of coelenterazine. Calculation of NF-κB activation in glioma-bearing mice treated with AAV-Vluc/sTRAIL or AAV-Vluc/GFP shown in f. Note that no Gluc signal was detected in the AAV-Vluc/GFP mice at any time point. Representative mice from each group are showing in (b,c, and e; n = 5/group). ITR, inverted terminal repeat; nd, not detected.

Figure 4

Effect of AAV-sTRAIL vector on U87 glioma tumors. (a,b) 5 × 10⁴ U87 glioma cells expressing Fluc, a marker for tumor volume, were implanted in the brain of nude mice. Fourteen days later, mice were randomized into two groups and infused into the same tumor injection site with either AAV-Vluc/GFP or AAV-Vluc/sTRAIL (n = 5/group). Mice were imaged for Fluc at days 2, 7, 14, and 21 after vector injection. (a) Representative set of mice from each group are shown. Note that the scale is different for each time point in order to show the tumor site. (b) Quantitation of brain tumor-associated Fluc signal in mice over time. The arrow indicates that all mice in the AAV-Vluc/GFP group were sacrificed between week 2 and week 3 due to tumor burden (*P = 0.048 at day14 and *P = 0.0008 at day 21). (c) Survival analysis of mice bearing U87 glioma tumors treated with either AAV-Vluc/GFP or AAV-Vluc/sTRAIL (n = 5; P = 0.0088). All Graphs are generated from a representative data set of three independent experiments.