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Journal Article

Bioware; PC-3M-luc; hVEGF-luc-PC3M

Background: Preclinical cancer studies increasingly utilize non-invasive imaging modalities. In the current study we have monitored tumor growth and vascular changes using two in vivo imaging tools: surface bioluminescence (BLI) and ultrasound biomicroscopy (UBM). BLI permits visualization of tumor location in the context of the whole body, including metastases localization. UBM imaging then permits high resolution 3D volumetric tumor measurements as well as blood flow estimates down to 200 microns/s. Measurements obtained from these complementary modalities were analyzed and compared to conventional, biochemical markers. Methods: Human prostate cancer cells expressing Firefly Luciferase constitutively (PC-3M-luc-C6) or under the control of hVEGF promoter (hVEGF-luc/PC3M) were implanted into male nude mice via an intradermal or subcutaneous injection. Tumor-bearing mice were subsequently imaged every week for nine weeks starting at week 2, by UBM to measure tumor burden using 3D volumetric analysis, or to estimate blood flow using speckle-variance flow processing. Surface bioluminescence was also acquired 10 minutes post i.p. injection of D-luciferin. In a longitudinal drug intervention study anti-hVEGF antibody (Bevacizumab, 200 ug) was injected i.p. into nude mice with subcutaneous xenografts of PC-3M-luc-C6 or hVEGF-luc/PC-3M twice per week for three weeks, starting at 14 days post-xenograft. UBM and surface BLI imaging were conducted every week. In order to study the correlation between VEGF expression in hVEGF-luc/PC3M xenografts (estimated by BLI) to tumor hypoxia level, mice were injected with pimonidazole hydrochloride (60 mg/kg i.v.) after three weeks of treatment and tumors were harvested for immunostaining analysis. Results: Surface BLI outputs (photons/s) from subcutaneous PC-3M-luc-C6 xenografts were highly correlated to tumor volumes measured using 3D UBM for small tumors (<100 mm3, r=0.92, n=8), yet poorly correlated to tumors of large size (>100 mm3, r=0.079, n=8). BLI signals in subcutaneous hVEGF-luc/PC3M xenografts showed an inverse trend to tumor blood flow. PC-3M-luc-C6 tumors treated with Bevacizumab showed growth inhibition by day 28 as demonstrated by 3D UBM (control vs treated = 67.27 vs 48.54 mm3). Moreover, control xenografts showed increased average BLI output over time, whereas treated tumors showed variation in BLI output. Necrosis, hypoxia and blood flow estimates were also investigated. Conclusions: Surface bioluminescence imaging demonstrated high correlations to accurate 3D UBM volumetric measurements of small tumor volumes, suggesting its usefulness in tracking early tumor growth quantitatively in drug intervention studies. A complementary imaging modality, like ultrasound biomicroscopy, is recommended to monitor tumor burden in advanced stages.

Journal Article

Androgens; Animals; Bioware; Cell Transformation, Neoplastic; Disease Models, Animal; Genes, Reporter; Humans; Image Processing, Computer-Assisted; In Situ Hybridization; Luciferases, Firefly; Luminescent Measurements; Male; Mice; Mice, Transgenic; PC-3M-luc; Promoter Regions, Genetic; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Journal Article

Animals; Bacterial Toxins; Biliary Tract; Bioware; Feces; Food Contamination; Intestines; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; pXen-5

Listeria monocytogenes is a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts. This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals, in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion of L. monocytogenes from the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen can occur very rapidly in the absence of fecal material. These studies demonstrate that L. monocytogenes bacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into the intestinal tract, where they pass into the environment and may possibly reinfect the animal.

Journal Article

Aminolevulinic Acid; Animals; Biofilms; Bioware; Cell Survival; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Implants, Experimental; Light; Luminescent Measurements; Methylene Blue; Osteomyelitis; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Sprague-Dawley; Staphylococcus aureus; Xen29

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1000 neonates and 1 in 5000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regimens of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (S. aureus) grown onto kirschner wires (K-wire). S. aureus-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of S. aureus biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. S. aureus infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg kg(-1)) intraperitoneally followed 4 h later by light (635 +/- 10 nm; 75 J cm(-2)) delivered transcutaneously via an optical fiber placed onto the tibia and resulted in significant delay in bacterial growth. In vitro, MB and ALA displayed similar cell kill with > or =4 log(10) cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat S. aureus cultures grown in vitro or in vivo using an animal model of osteomyelitis.

Journal Article

Acetates; Alginates; Animals; Anti-Infective Agents; Bandages; Bioware; Chitosan; Glucuronic Acid; Hexuronic Acids; Male; Mice; Mice, Inbred BALB C; Occlusive Dressings; Proteus mirabilis; Pseudomonas aeruginosa; Silver Sulfadiazine; Staphylococcus aureus; Wound Healing; Wound Infection; Xen8.1, Xen5, Xen44

HemCon bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50-250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25-100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.