Christian E. Badr

A secreted luciferase for ex-vivo monitoring of in vivo processes

Luciferases are widely used to monitor biological processes. Here we describe the naturally secreted Gaussia princeps luciferase (Gluc) as a highly sensitive reporter for quantitative assessment of cells in vivo by measuring its concentration 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.

Bioluminescence imaging: progress and applications

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

Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

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 tumour 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 labelling. 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 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

Background The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.

Real-time monitoring of nuclear factor kappaB activity in cultured cells and in animal models.

Nuclear factor κB (NF-κB) is a transcription factor that plays a major role in many human disorders, including immune diseases and cancer. We designed a reporter system based on NF-κB responsive promoter elements driving expression of the secreted Gaussia princeps luciferase (Gluc). We show that this bioluminescent reporter is a highly sensitive tool for noninvasive monitoring of the kinetics of NF-κB activation and inhibition over time, both in conditioned medium of cultured cells and in the blood and urine of animals. NF-κB activation was successfully monitored in real time in endothelial cells in response to tumor angiogenic signaling, as well as in monocytes in response to inflammation. Further, we demonstrated dual blood monitoring of both NF-κB activation during tumor development as correlated to tumor formation using the NF-κB Gluc reporter, as well as the secreted alkaline phosphatase reporter. This NF-κB reporter system provides a powerful tool for monitoring NF-κB activity in real time in vitro and in vivo.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

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.

Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway

Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.

Functional drug screening assay reveals potential glioma therapeutics.

Here we describe a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells. Using this assay, we identified small-molecule drugs that sensitized brain tumor cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. Human glioblastoma multiforme cells were engineered by gene transfer to express Gluc as a reporter for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay for screening and validated it using the National Institute of Neurological Disorders and Stroke (NINDS) custom collection II library consisting of 1,040 drugs and bioactive compounds, most of which are Food and Drug Administration-approved and are able to cross the blood-brain barrier. We found that the cardiac glycosides family sensitized glioblastoma multiforme cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. In conclusion, the Gluc secretion assay is a robust tool for functional drug screening and can be applied to many different fields including cancer.

Bioluminescence Imaging: Basics and Practical Limitations

Over the last three decades, imaging has been a thriving field with continuous egression of more reliable and highly sophisticated tools and techniques allowing better understanding of biological processes in living organisms. This field continues to expand and its applications broaden to encompass limitless applications in various biomedical research areas. It is however, of utmost importance to understand the capabilities and limitations of this technique as new challenges and hurdles continue to arise. This chapter describes the general properties of bioluminescence imaging and commonly used reporters while underlining the challenges and limitations with these modalities.

Systemic Anticancer Neural Stem Cells in Combination with a Cardiac Glycoside for Glioblastoma Therapy

The tumor‐tropic properties of neural stem cells (NSCs) have been shown to serve as a novel strategy to deliver therapeutic genes to tumors. Recently, we have reported that the cardiac glycoside lanatoside C (Lan C) sensitizes glioma cells to the anticancer agent tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Here, we engineered an FDA‐approved human NSC line to synthesize and secrete TRAIL and the Gaussia luciferase (Gluc) blood reporter. We showed that upon systemic injection, these cells selectively migrate toward tumors in the mice brain across the blood‐brain barrier, target invasive glioma stem‐like cells, and induce tumor regression when combined with Lan C. Gluc blood assay revealed that 30% of NSCs survived 1 day postsystemic injection and around 0.5% of these cells remained viable after 5 weeks in glioma‐bearing mice. This study demonstrates the potential of systemic injection of NSCs to deliver anticancer agents, such as TRAIL, which yields glioma regression when combined with Lan C. Stem Cells 2014;32:2021–2032