Bioluminescence Monitoring Research Articles

A Single-Cell Bioluminescence Imaging System for Monitoring Cellular Gene Expression in a Plant Body

Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Abstract 1091: Procoagulant receptor tissue factor provokes the escape from brain tumor dormancy.

Abstract Occult persistence of transformed cells is known to precede the onset of clinically apparent malignancies, accompany disease remission and micrometastasis. While the mechanisms governing this dormant state remain poorly understood, tissue injury and wound healing responses have often been implicated in its cessation, but the surrounding circumstances remain largely obscure. Notably, the activation of hemostasis represents the first response to the exogenous and endogenous tissue injury, and is linked to inflammation, angiogenesis and growth promoting microenvironment. Tissue factor (TF) acts as the main trigger of the coagulation cascade in this setting, and is known to be overexpressed in cancer cells, including in high grade brain tumors such as glioblastoma (GBM). Here we report that in a model of GBM, the TF-mediated activation of the coagulation system may act as the sufficient initial trigger in the cessation of tumor dormancy, and the onset of inflammation and angiogenesis. We also document that while TF may play a role in the subsequent tumor progression, its expression by cancer and host cells may no longer be absolutely required in established tumors. Thus, we observed that indolent human glioma cells (U373) remain viable, but dormant for over 250 days post subcutaneous and orthotopic inoculation, with no evidence of tumor growth, as detected by bioluminescent monitoring. The enforced expression of TF is sufficient to trigger tumor formation by these cells, albeit after long latency (30-90 days). During this latent phase TF-expressing glioma cells, but not their TF-negative counterparts, are able to recruit rich stroma, containing CD11b+ myeloid cells and CD105+ blood vessels. These cells also evolve to become rapidly tumorigenic and gain a dramatic change to their transcriptome. Similar aggressiveness can also be induced in U373 cells by the expression of the oncogenic epidermal growth factor receptor variant (EGFRvIII), which also provokes upregulation of TF and onset of the procoagulant phenotype (U373vIII cells). Targeting TF in U373vIII tumors, as well as their implantation into TF-hypomorphic recipients (low-TF mice) delays, but does not abrogate experimental GBM progression. We generated spontaneous GBM tumors by intracranial injection of the oncogenic cDNA (SV40, N-ras), which becomes expressed in brain astrocytes under the control of the Sleeping Beauty (SB) transposase. Disruption of the TF gene in the astrocytic lineage of such mice (Cre/nestinTF-/-) resulted in a partial tumor growth inhibition. We postulate that activation of the coagulation system (via TF) may play a role in disruption of the dormant behavior of brain tumor cells and can serve as targets in prevention of disease onset. However, targeting TF alone is only partially effective as a measure to control the growth of established astrocytic tumors. Acknowledgments: We thank Dr. John Ohlfest for providing us with the SB transposon system. Citation Format: Nathalie Magnus, Delphine Garnier, Brian Meehan, Maryam Hashemi, Tae-Hoon Lee, Chloe Milsom, Nada Jabado, Rafal Pawlinski, Nigel Mackman, Janusz Rak. Procoagulant receptor tissue factor provokes the escape from brain tumor dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1091. doi:10.1158/1538-7445.AM2013-1091

Abstract 402: A new mathematical model for describing metastatic spreading: Validation in tumor-bearing mice, confrontation with clinical data and in silico simulations to optimize treatment modalities.

Abstract Occult metastatic disease is a major concern in clinical oncology because optimal therapy can not been undertaken in a timely manner. Developing mathematical tools for describing and anticipating early metastatic stages would help clinicians to choose the most adequate therapeutic strategy, even if no metastasis is yet detectable at bedside. We have developed a mathematical model that provides a Metastatic Index (MI). We used a phenomenological approach based on a structured transport equation with non local boundary condition for the colony size distribution of metastases. The velocity of this transport was related to a Gompertz law's growth. The colonization rate of the tumors reflects not only the metastatic diffusion but also some fractal dimension of the blood vessels infiltrating the tumors. This model is mostly based upon few parameters and can integrate the impact of surgery or chemotherapies on tumor growth and spreading. Model structure and parameters were first adjusted by fitting the predictions with observed data from several experiments performed in mice bearing the MDA-231LUC+ breast orthotopic xenograft. Three-dimmensional bioluminescence monitoring was carried out so as to detect early metastases as small as 10ˆ5 cells. A pre-validation step was carried out to check the ability of bioluminescence imaging to discriminate small metastatic sites from artifactual signals. Various molecular markers (A-cadherine, alcohol dehydrogenase, metaloproteases) were studied from tumor biopsies to refine some of the model parameters. Data were finally compared using Monolix software and Visual Predictive Check confirmed the ability of the model to predict accuratelly both tumor growth-rate and invasiveness. Additionally, we compared the in silico predictions of the model with clinical data from 2648 breast cancer patients (a.k.a. the Koscielny cohort) with a follow-up of metastatic reccurence depending on the initial tumor size measured upon surgery. Results showed that the model's predictions matched the clinical observations (rˆ2=0.98), thus suggesting that our MI could be a useful tool indeed to forecast metastatic spreading in patients with cancer. Finally, in silico simulations were performed to study the impact of surgery or treatment with cytotoxics alone or combined with antiangiogenics. Marked variations in efficacy were observed depending on the treatment modalities. The resulting simulations suggest therefore that our mathematical model could be used as well to determine in silico the best scheduling and dosing of cancer chemotherapy, especially when anti-angiogenic and cytotoxic drugs are associated. Citation Format: Severine Mollard, Joseph Ciccolini, Niklas Hartung, Christian Faivre, Sébastien Benzekri, Guillemette Chapuisat, Assia Benabdallah, Florence Hubert, Dominique Barbolosi. A new mathematical model for describing metastatic spreading: Validation in tumor-bearing mice, confrontation with clinical data and in silico simulations to optimize treatment modalities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2013-402

Real-Time Analysis of the Circadian Oscillation of the Rev-Erb ^|^beta; Promoter

Rev-Erb β gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb β that generate and determine the phase of the circadian oscillation remain unclear. We analyzed the Rev-Erb β promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays. Luciferase reporter assays indicated that only the 5' region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb β oscillation. Based on EMSA results, REV-ERBβ binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity. We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb β expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb β oscillation phase. Our results offer new insight into the role of Rev-Erb β in the circadian rhythm system.

Temporal and spatial distribution of murine placental and brain GLUT3-luciferase transgene as a readout of in vivo transcription

To investigate in vivo transcription of the facilitative glucose transporter isoform-GLUT3 gene, we created GLUT3-firefly luciferase transgenic mouse lines that demonstrate tissue-specific [adult: brain > testis ≥ skeletal muscle > placenta; postnatal (PN): skeletal muscle > brain = skin], temporal, and spatial distribution of the reporter gene/enzyme activity that is unique from endogenous GLUT3 mRNA/protein. In this mouse model, luciferase expression/activity serving as a readout of in vivo transcription peaked at 12 days gestation along with proliferating cell nuclear antigen (cell replication) in placenta and embryonic brain preceding peak GLUT3 protein expression at 18-19 days gestation. In contrast, a postnatal increase in brain luciferase mRNA peaked with endogenous GLUT3 mRNA, but after that of NeuroD6 protein (neurogenesis) at PN7. Luciferase activity paralleled GLUT3 protein expression with Na(+)-K(+)-ATPase (membrane expansion) and synaptophysin (synaptogenesis) proteins, peaking at PN14 and lasting until 60 days in the adult. Thus GLUT3 transcription in placenta and embryonic brain coincided with cell proliferation and in postnatal brain with synaptogenesis. Longitudinal noninvasive bioluminescence (BLI) monitoring of in vivo brain GLUT3 transcription reflected cross-sectional ex vivo brain luciferase activity only between PN7 and PN21. Hypoxia/reoxygenation at PN7 revealed transcriptional increase in brain GLUT3 expression reflected by in vivo BLI and ex vivo luciferase activity. These observations collectively support a temporal contribution by transcription toward ensuring adequate tissue-specific, developmental (placenta and embryonic brain), and postnatal hypoxic brain GLUT3 expression.

IL-1 Generated Subsequent to Radiation-induced Tissue Injury Contributes to the Pathogenesis of Radiodermatitis

IL-1 Generated Subsequent to Radiation-induced Tissue Injury Contributes to the Pathogenesis of Radiodermatitis

SU-E-J-198: Bioluminescence Monitoring of Metastatic Breast Cancer: Quantitative Assessment of Radiation Treatment Effects and Tracking of Tumor Cells.

To evaluate radiation treatment effects on mammary carcinoma cells, quantitative photon radiance were monitored to track light-emitting cancer cells and metastasis using in vivo bioluminescence imaging. Eight female BALB/c mice aged 8 weeks were orthotopically injected with 5×104/cc 4T1 tumor cells into the abdominal mammary gland. The firefly luciferase-based bioluminescence images were acquired every 2-3 days for 1 month. Bioluminescent intensity was analyzed in average surface radiance (photons/sec/cm2 /sr) taken in 3-dimensional bioluminescence tomography (BLT). After 1 week, single-radiation dose of 20 Gy was delivered by orthovoltage X-rays. Variation of detected bioluminescence signals emitted from molecular cancer cells was depicted on BLT images. To delineate tumor volumes according to bioluminescence intensity on anatomical images for radiation therapy, BLT images were registered with the micro computed tomography (CT) images using surface-constrained warping. Multispectral BLT images elaborated on early detection of cancer cells, characteristics of tumor growth, and metastasis for more accurate determination of internal bioluminescent sources. The radiation-treated mice having only primary tumor volumes showed 67% decrease in bioluminescent signals, while the mice with metastatic cancer cells suggested 88% reduction, as compared to the control group. Registration of BLT with CT images guided molecular cancer cells on anatomical coordinates. The BLT imaging was a useful tool to localize cancer cells and to quantify radiation response. Application of BLT led to more accurate definition of tumor volumes including molecular probe-based microscopic cancer cells. Monitoring of bioluminescence signals enables to diagnose real-time metastatic behavior of cancer cells and determine optimal radiation treatment strategies adapted to tumor characteristics.

Abstract 5718: Implementation of μ-Image Guided Radio-Therapy in the treatment of experimental glioma mouse models: Assessment of the potential of agents that interfere with DNA repair

Abstract High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. In order to evaluate the potential of novel therapies, we have developed a set of glioma mouse models. These include LoxP conditional mouse models that form spontaneous high-grade gliomas following intracranial injection of lentiviral CMV-Cre vectors and high-grade glioma models that develop following intracranial injection of cell lines obtained from these spontaneous models and maintained as neurosphere cultures. Standard treatment of newly diagnosed high-grade glioma after surgical resection involves radiotherapy (RT) and chemotherapy (CT) with temozolomide. Both modalities are based on inflicting damage to the DNA of tumor cells but have only a limited effect on overall survival. A potential strategy to improve the efficacy of DNA damaging therapies is to combine these with agents that interfere with DNA damage repair. In this study we have concentrated on the inhibition of PARP by ABT-888 and the inhibition of Wee1 kinase by PD0166285 and MK-1775. Importantly, we have mimicked the therapy of patients as closely as possible by implementing μ-Image Guided Radiotherapy (μ-IGRT) using the X-Rad 225Cx (Precision X-Ray Inc). Through cone beam CT guidance this system offers precise delivery of high energy beams (225 KVp) of small field sizes (1 - 5 mm), minimizing the exposure of normal tissues and allowing the delivery of RT doses that can not be given by conventional whole body RT. Here RT was delivered using a fractionated schedule (5 Gy per day x 4) in combination with oral temozolomide (100 mg/kg/day x 4) alone or with ABT-888 (10 mg/kg/bid x 4), PD0166285 (0.25 mg/kg/bid x 4 or MK1775 (20 mg/kg/bid x 4). Treatment of orthotopically injected Ink4a/Arf;P53;K-Rasv12 neurosphere-cultured cells (GBM652457) by RT + CT was much more efficacious than by CT alone. In line with the expectations, the PARP inhibitor ABT-888 significantly improved the response (assessed by bioluminescence monitoring) nd survival. However, the same combination was not more efficacious against spontaneous Ink4a/Arf;P53;K-Rasv12 tumors relative to RT + CT alone. Addition of the Wee1 kinase inhibitors PD0166285 or MK1775 did not improve the efficacy of RT+CT against intracranially injected GBM652457 cells. We are currently investigating the underlying reason of these results. ABT-888, PD0166285 and MK1775 are all substrates of ABCB1 and ABCG2 and especially MK1775 has a very poor BBB penetration. In conclusion, μ-IGRT is an exciting new technique to mimic treatment of glioma patients in mouse models more closely. Since all novel therapies for treatment of high-grade glioma will be given in conjunction to the current chemoradiation therapy, this technique will allow more accurate preclinical evaluation of novel therapies in a clinically relevant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5718. doi:1538-7445.AM2012-5718

Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis

The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca2+ oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion.

Salicylic acid-releasing polyurethane acrylate polymers as anti-biofilm urological catheter coatings

Biofilm-associated infections are a major complication of implanted and indwelling medical devices like urological and venous catheters. They commonly persist even in the presence of an oral or intravenous antibiotic regimen, often resulting in chronic illness. We have developed a new approach to inhibiting biofilm growth on synthetic materials through controlled release of salicylic acid from a polymeric coating. Herein we report the synthesis and testing of a ultraviolet-cured polyurethane acrylate polymer composed, in part, of salicyl acrylate, which hydrolyzes upon exposure to aqueous conditions, releasing salicylic acid while leaving the polymer backbone intact. The salicylic acid release rate was tuned by adjusting the polymer composition. Anti-biofilm performance of the coatings was assessed under several biofilm forming conditions using a novel combination of the MBEC Assay™ biofilm multi-peg growth system and bioluminescence monitoring for live cell quantification. Films of the salicylic acid-releasing polymers were found to inhibit biofilm formation, as shown by bioluminescent and GFP reporter strains of Pseudomonas aeruginosa and Escherichia coli. Urinary catheters coated on their inner lumens with the salicylic acid-releasing polymer significantly reduced biofilm formation by E. coli for up to 5days under conditions that simulated physiological urine flow.

Lentiviral Transduction of Patient Derived Leukaemic Blasts Allowing In Vivo Bioluminescent Monitoring in An NSG Model of Leukaemia Stem Cell Maintenance,

Abstract 4000Serial transplantation of patient derived acute lymphoblastic leukaemia (ALL) blasts continues to contribute to our understanding of the biology of leukaemia stem cells (LSC). Refinement of techniques, and in particular intrafemoral injection and development of the highly immunocompromised NOD/scid IL2Rγ null (NSG) mouse have demonstrated B precursor ALL propagating cells to be both common and present in diverse immunophenotypes. These studies must now be complemented by interrogation of the biological pathways underpinning leukaemia stem cell behaviour and clonal propagation in ALL. We have developed a lentiviral based approach to such studies, using the transfer vector pSLIEW, encoding both enhanced green fluorescent protein (EGFP) and firefly luciferase (luc).We have recently replaced a bone marrow stromal feeder based transduction protocol with a feeder free protocol, removing the risk of co-transduction of feeder cells. Using the feeder free protocol, we have achieved transduction of primary (n=4) and primograft (n=3) material with between 13.0 and 51.4% eGFP positive cells.Transplantation of transduced cells by intrafemoral injection into NSG mice resulted in engraftment and disease dissemination. This process was monitored using an IVIS Spectrum bioluminescence imaging system. This technique demonstrated progression of disease to the contralateral femur, spleen, CNS and vertebrae. Disease progression was also monitored by serial bone marrow punctures and 5-colour flow cytometry, which demonstrated no immunophenotypic bias amongst the transduced cells. Flow cytometry of harvested bone marrow and spleen showed between 5.5% and 10.2% eGFP positive cells representing only a moderate decrease from 26.3% eGFP positivity at initial transplantation. This confirms the relative resistance of the SFFV promoter to silencing, making this approach suitable for serial transplantation.Harvested bone marrow and splenic cells were re-transplanted at 5.5 × 103 – 1.0 × 104 SLIEW+ cells per mouse (total 1 × 105 cells transplanted). Bioluminescent imaging has shown engraftment and dissemination of leukaemia within five weeks, confirming transduction of the leukaemia stem cell compartment.Further development of the pSLIEW vector to include shRNA sequences now offers the potential for functional studies using patient derived material, transduced with a single lentivector construct and serially engrafted in the NSG assay for leukaemic stem cell maintenance. We believe that this approach will allow us to investigate the genetic programmes underpinning leukaemia stem cell self-renewal. Disclosures:No relevant conflicts of interest to declare.

Synergistic Combination of Chitosan Acetate with Nanoparticle Silver as a Topical Antimicrobial: Efficacy against Bacterial Burn Infections

Chitosan and nanoparticle silver are both materials with demonstrated antimicrobial properties and have been proposed singly or in combination as constituents of antimicrobial burn dressings. Here, we show that they combine synergistically to inhibit the in vitro growth of Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria (Pseudomonas aeruginosa, Proteus mirabilis, and Acinetobacter baumannii), as judged by bioluminescence monitoring and isobolographic analysis, and also produce synergistic killing after 30 min of incubation, as measured by a CFU assay. The hypothesized explanation involves chitosan-mediated permeabilization of bacterial cells, allowing better penetration of silver ions into the cell. A dressing composed of freeze-dried chitosan acetate incorporating nanoparticle silver was compared with a dressing of chitosan acetate alone in an in vivo burn model infected with bioluminescent P. aeruginosa. The survival rates of mice treated with silver-chitosan or regular chitosan or left untreated were 64.3% (P = 0.0082 versus regular chitosan and P = 0.0003 versus the control), 21.4%, and 0%, respectively. Most of the fatalities occurred between 2 and 5 days postinfection. Silver-chitosan dressings effectively controlled the development of systemic sepsis, as shown by blood culture. These data suggest that a dressing combining chitosan acetate with silver leads to improved antimicrobial efficacy against fatal burn infections.

Abstract 5253: A model of tumor dormancy in Luminal A breast cancer

Abstract Introduction: The clinical course and biologic features of metastasis vary extensively depending on the type of cancer. Latency is one of the most important variables in metastasis. Late-onset recurrences are frequently found in Luminal A (ER+/PR+/Her2-) breast cancer patients. In both ER+ tumors and ER+ breast cancer cell lines, tumor cells preferentially disseminate to the bone marrow where they are capable of remaining dormant. The identification of mechanisms that support the survival of disseminated cancer cells (DTCs) has clear implications for understanding the biology of latent metastatic disease. Method: ER+ breast cancer cell lines expressing a GFP luciferase vector (for in vivo bioluminescent monitoring of tumor growth and dissemination) were orthotopically injected in the mammary fat pad (MFP) of mice. DTCs were isolated and characterized from the bone marrow and lungs after several months. Results: The isolated DTCs from the bone and lung do not proliferate, are resistant to both chemo and hormonal therapy (HT) and are endowed with a highly invasive/disseminating capacity (loss of E-Cadherin and gain of EMT features). Moreover, these DTCs exhibit decreased expression of ER compared to the cancer cells within the primary tumor. Since ER activity is required for ER+ breast cancer cell growth in vitro and in vivo, our data suggest that loss of ER activity can be a mechanism of tumor dormancy in Luminal A breast cancer. Conclusion: An understanding of the consequences of ER activity in terms of tumor dormancy and dissemination potential of ER+ breast cancer cells has implications for improving the treatment of latent metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2011-5253

Effect of humic substances on toxicity of inorganic oxidizer bioluminescent monitoring

The current study deals with the effect of humic substances (HS) on toxicity of solutions of a model inorganic oxidizer, potassium ferricyanide. Chemical reactions responsible for toxicity changes are under consideration. The bioluminescent system of coupled enzymatic reactions catalyzed by bacterial luciferase and oxidoreductase was used as a bioassay. General and oxidative toxicity of ferricyanide solutions were evaluated. Ability of HS to decrease or increase general and oxidative toxicity of the solutions was revealed. Two types of chemical processes are supposed to be responsible for detoxification by HS: ferricyanide-HS complex formation and acceleration of endogenous redox reactions in the bioluminescent assay system. Decrease of oxidative toxicity of ferricyanide solution was observed under incubation with HS at all concentrations of HS used. Conditions for general toxicity decrease were prior incubation of ferricyanide with HS and low HS concentrations (< 10⁻⁴g/L). Acceleration of NADH auto-oxidation under higher HS concentrations was supposed to result in a toxicity increase.

In vivo bioluminescence monitoring of defense gene expression in response to treatment with yeast cell wall extract

Previous studies indicated that Housaku Monogatari (HM), a plant activator made from yeast cell wall extract, is effective for control of various plant diseases. To investigate the effect of HM treatment on plant gene expression, we tested the expression of defense related gene induction by exploiting tobacco pathogenesis-related protein 1a (PR-1a) gene promoter- and the Arabidopsis plant defensin 1.2 (PDF1.2) gene promoter-luciferase fusions as reporter genes. Transgenic Arabidopsis plants harboring promoter-luciferase fusion genes were treated with HM and the promoter activity was monitored as changes in luciferase activity in planta. Results of bioluminescence monitoring assay indicated that the promoters were activated at different times after the treatment of test plants with HM. Maximum activation of the PR-1a promoter occurred 4 days, and of the PDF1.2 promoter 4 h, after treatment. These results suggest that HM might contain multiple microbe-associated molecular patterns (MAMPs) that activate systemic acquired resistance and induced systemic resistance signaling pathways at different times. This may explain the mechanisms involved in the induction of defense responses against multiple plant pathogens by HM treatment.

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.

Detoxification of AM-241 solutions by humic substances: bioluminescent monitoring

The study addresses the effect of humic substances on marine luminous bacteria Photobacterium phosphoreum exposed to Am-241 (3,000BqL(-1), water solution). Luminescent intensity of the bacteria was applied as a marker of their physiological activity. Humic substances have been found to reduce the effect of Am-241 on luminescence, decrease damage to cells, and change distribution of Am-241 between bacterial cells and intercellular media. It was shown that water-soluble humic substances, being products of natural transformation of organic substances in soil and bottom sediments, can serve as protecting agents for water microorganisms exposed to alpha radionuclides.

Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment In a Humanized Mouse Model

AbstractAbstract 1927Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress. Disclosures:Jamieson:Bristol-Meyers Squibb: Research Funding.

Inhibition of the Inflammatory Cytokine TNF-α Increases Adenovirus Activity in Ovarian Cancer via Modulation of cIAP1/2 Expression

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.