Serum Amyloid A3 Promoter-Driven Luciferase Activity Enables Visualization of Diabetic Kidney Disease

The early detection of diabetic nephropathy (DN) in mice is necessary for the development of drugs and functional foods. The purpose of this study was to identify genes that are significantly upregulated in the early stage of DN progression and develop a novel model to non-invasively monitor disease progression within living animals using in vivo imaging technology. Streptozotocin (STZ) treatment has been widely used as a DN model; however, it also exhibits direct cytotoxicity to the kidneys. As it is important to distinguish between DN-related and STZ-induced nephropathy, in this study, we compared renal responses induced by the diabetic milieu with two types of STZ models: multiple low-dose STZ injections with a high-fat diet and two moderate-dose STZ injections to induce DN. We found 221 genes whose expression was significantly altered during DN development in both models and identified serum amyloid A3 (Saa3) as a candidate gene. Next, we applied the Saa3 promoter-driven luciferase reporter (Saa3-promoter luc mice) to these two STZ models and performed in vivo bioluminescent imaging to monitor the progression of renal pathology. In this study, to further exclude the possibility that the in vivo bioluminescence signal is related to renal cytotoxicity by STZ treatment, we injected insulin into Saa3-promoter luc mice and showed that insulin treatment could downregulate renal inflammatory responses with a decreased signal intensity of in vivo bioluminescence imaging. These results strongly suggest that Saa3 promoter activity is a potent non-invasive indicator that can be used to monitor DN progression and explore therapeutic agents and functional foods.

Quantification of bacteria by in vivo bioluminescence imaging in comparison with standard spread plate method and reverse transcription quantitative PCR (RT-qPCR)

In vivo bioluminescence imaging (BLI) offers a unique opportunity to analyze ongoing bacterial infections qualitatively and quantitatively in intact animals over time, leading to a reduction in the number of animals needed for a study. Since accurate determination of the bacterial burden plays an essential role in microbiological research, the present study aimed to evaluate the ability to quantify bacteria by non-invasive BLI technique in comparison to standard spread plate method and reverse transcription quantitative PCR (RT-qPCR). For this purpose, BALB/c mice were intranasally infected with 1 × 105 CFU of bioluminescent Streptococcus pneumoniae A66.1. At day 1 post-infection, the presence of S. pneumoniae in lungs was demonstrated by spread plate method and RT-qPCR, but not by in vivo BLI. However, on the second day p.i., the bioluminescent signal was already detectable, and the photon flux values positively correlated with CFU counts and RT-qPCR data within days 2–6. Though in vivo BLI is valuable research tool allowing the continuous monitoring and quantification of pneumococcal infection in living mice, it should be kept in mind that early in the infection, depending on the infective dose, the bioluminescent signal may be below the detection limit.

A novel thermostable beetle luciferase based cytotoxicity assay

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed ‘Matador-Glo assay’ which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

Near-Infrared Luciferin Analogs for In Vivo Optical Imaging

The firefly bioluminescence reaction has been exploited for in vivo optical imaging in life sciences. To develop highly sensitive bioluminescence imaging technology, many researchers have synthesized luciferin analogs and luciferase mutants. This chapter first discusses synthetic luciferin analogs and their structure–activity relationships at the luminescence wavelength of the firefly bioluminescence reaction. We then discuss the development of luciferin analogs that produce near-infrared (NIR) light. Since NIR light is highly permeable for biological tissues, NIR luciferin analogs might sensitively detect signals from deep biological tissues such as the brain and lungs. Finally, we introduce two NIR luciferin analogs (TokeOni and seMpai) and a newly developed bioluminescence imaging system (AkaBLI). TokeOni can detect single-cell signals in mouse tissue and luminescence signals from marmoset brain, whereas seMpai can detect breast cancer micro-metastasis. Both reagents are valid for in vivo bioluminescence imaging with high sensitivity.

How to Select Firefly Luciferin Analogues for In Vivo Imaging

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.

Abstract 13143: Safety Evaluation of Therapeutic Angiogenesis With Adipose-derived Regenerative Cells for Ischemic Limb in a Tumor Bearing Model

Introduction: Implantation of adipose-derived regenerative cells (ADRC) is a promising novel strategy to augment angiogenesis and blood perfusion recovery in ischemic diseases with no other therapeutic option. However, there is a clinical concern underlying therapeutic angiogenesis that implantation of ADRC may promote tumor growth and metastasis via remote angiogenesis. Accordingly, we tested whether therapeutic angiogenesis with ADRC against hindlimb ischemia (HLI) would affect remote tumor growth and angiogenesis in a tumor-bearing mouse ischemic hindlimb model. Methods and results: B16F10-Luc (murine melanoma cells expressing luciferase, 1x106 cells/animal) were implanted to C57BL/6J mice’s (male, 8-10 weeks old, n=10) back. Mice were subjected to unilateral HLI surgery one day after tumor implantation. Then, mice were randomly assigned to the control group or the ADRC group (n=5 for each). ADRC (1x106 cells/animal) or PBS were implanted/injected into ischemic hindlimb muscles one day after the surgery. Blood perfusion recovery in HLI by laser Doppler perfusion imaging system and tumor size by a caliper were measured every week up to 21 days after surgery. At POD 21, tumor weight and luciferase activity in primary tumors obtained by in vivo bioluminescence imaging system were also evaluated. Immunohistochemistry by CD31 or LYVE1 staining was performed to detect feeder arteries or outflow lymphatic vessels in tumors. The results demonstrated that better blood perfusion recovery and more capillary density in HLI was observed in the ADRC group than in the control group (p<0.05, respectively). However, there were no significant differences in terms of tumor volume (p=0.95), tumor weight (p=0.88) and luciferase activity of primary tumor (p=0.92) between those two groups. No sign of distant metastasis was detected by macroscopic and pathological examination, and by in vivo bioluminescence imaging system in both groups. Further study also revealed that capillary density of peritumoral blood vessels or lymphatic vessels was not augmented by ADRC implantation into remote HLI. Conclusions: Our data indicated that therapeutic angiogenesis with ADRC implantation against HLI did not promote remote tumor growth, angiogenesis and metastasis.

INNV-13. SYNERGISTIC EFFECT OF COLD ATMOSPHERIC PLASMA IN COMBINATION WITH TEMOZOLOMIDE TO TREAT GLIOBLASTOMA IN A NON-INVASIVE MOUSE XENOGRAFT MODEL

INTRODUCTION A primary limitation in anti-cancer therapy is the resistance of cancer cells to chemotherapeutic drugs. However, combination therapy may be an effective approach for reducing drug derived toxicity and evading drug resistance, resulting in improved clinical treatment of cancer. Our prior work demonstrated effective treatment of glioblastoma (GBM) with cold atmospheric plasma (CAP) technology with minimal effect to normal cells. Consequently, CAP may serve as a strong candidate for combination therapy with the classical antineoplastic alkylating agent Temozolomide (TMZ) to treat GBM. OBJECTIVES To determine the in vivo co-efficacy of CAP and TMZ to “sensitize” GBM. METHODS An in vivo study was performed using the CAP jet device (He-gas) to determine the effect of combined CAP–TMZ treatment. U87MG-luc glioblastoma cells were implanted intracranially in athymic nude NU(NCr)-Foxn1nu/immunodeficient mice. He-CAP (or control He alone) was non-invasively applied over the skin for 60sec to developed tumors on the first day of the treatment followed with 6.5 mg/kg TMZ or vehicle control treatment for 5 days for two weeks (n=5/group). In vivo bioluminescence imaging was used to monitor tumor volume on the 6th, 9th and 13th treatment day. RESULTS In vivo bioluminescence imaging revealed a marked 8.0±3.2 fold increase in tumor volume in control animals (He-vehicle). Treatment with He-TMZ (6.7±2.5 fold) or CAP-vehicle (4.8±1.7 fold) in isolation had minimal effect in preventing tumor growth. However, combined CAP-TMZ co-treatment virtually prevented increases in tumor volume over 2 weeks (1.8±0.2 fold). CONCLUSIONS Collectively, these findings indicate an effective synergistic treatment method for GBM combining CAP with TMZ. Future investigations look to incorporate radiation into the treatment regimen as well as primary GBM cell models.

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid, direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission

In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase–luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is necessary for in vivo animal experiments. Various attempts have been undertaken to achieve this goal, including genetic engineering of luciferase, chemical modulation of luciferin, and utilization of bioluminescence resonance energy transfer (BRET). In this review, we overview a recent advance in the development of a bioluminescence system for in vivo BLI. We also specifically examine the improvement in bioluminescence intensity by mutagenic or chemical modulation on several beetle and marine luciferase bioluminescence systems. We further describe that intramolecular BRET enhances luminescence emission, with recent attempts for the development of red-shifted bioluminescence system, showing great potency in in vivo BLI. Perspectives for future improvement of bioluminescence systems are discussed.