Journal Article

AngioSense, IVIS, Alphavirus Infections/pathology/*therapy/virology; Animals; Antineoplastic Agents, Phytogenic/therapeutic use; Blotting, Western; Cell Membrane Permeability; Combined Modality Therapy; Cricetinae; Drug Delivery Systems; Female; *Genetic Vectors; Humans; Mice; Mice, SCID; Neovascularization, Pathologic/*prevention & control; Neuroblastoma/blood supply/therapy/virology; *Oncolytic Virotherapy; Ovarian Neoplasms/*blood supply/*therapy/virology; Paclitaxel/therapeutic use; Sindbis Virus/*physiology; Vascular Endothelial Growth Factor A/metabolism; Xenograft Model Antitumor Assays

Genetic instability of cancer cells generates resistance after initial responses to chemotherapeutic agents. Several oncolytic viruses have been designed to exploit specific signatures of cancer cells, such as important surface markers or pivotal signaling pathways for selective replication. It is less likely for cancer cells to develop resistance given that mutations in these cancer signatures would negatively impact tumor growth and survival. However, as oncolytic viral vectors are large particles, they suffer from inefficient extravasation from tumor blood vessels. Their ability to reach cancer cells is an important consideration in achieving specific oncolytic targeting and potential vector replication. Our previous studies indicated that the Sindbis viral vectors target tumor cells by the laminin receptor. Here, we present evidence that modulating tumor vascular leakiness, using VEGF and/or metronomic chemotherapy regimens, significantly enhances tumor vascular permeability and directly enhances oncolytic Sindbis vector targeting in tumor models. Because host-derived vascular endothelium cells are genetically stable and less likely to develop resistance to chemotherapeutics, a combined metronomic chemotherapeutics and oncolytic vector regimen should provide a new approach for cancer therapy. This mechanism could explain the synergistic treatment outcomes observed in clinical trials of combined therapies.

Journal Article

animal cell, animal model, article, bacterial colonization, bacterial growth, bacterial virulence, bioluminescence, cell culture, controlled study, extracellular space, gallbladder, in vivo study, Listeria monocytogenes, mouse, nonhuman, priority journal IVIS, Xenogen, Xen32

The bacterium Listeria monocytogenes can cause a life-threatening systemic illness in humans. Despite decades of progress in animal models of listeriosis, much remains unknown about the processes of infection and colonization. Here, we report that L. monocytogenes can replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal. In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals. The data suggest that L. monocytogenes may be carried in the human gall bladder.

Journal Article

Animals, Anti-Bacterial Agents/ pharmacology, Bacterial Infections/drug therapy/microbiology, Biofilms/ drug effects/growth & development, Bioware; Catheterization/adverse effects, Chemiluminescent Measurements, Ciprofloxacin/pharmacology, Colony Count, Microbial, Disease Models, Animal, Dose-Response Relationship, Drug, Drug Monitoring/methods, Mice, Rifampin/pharmacology, Staphylococcus aureus/drug effects/genetics/growth & development, Tobramycin/pharmacology IVIS, Xenogen; Xen29

We have developed a rapid, continuous method for monitoring the effectiveness of several antibacterial agents in real time, noninvasively, by using a recently described mouse model of chronic biofilm infection (J. L. Kadurugamuwa et al., Infect. Immun. 71:882-890, 2003), which relies on biophotonic imaging of bioluminescent bacteria. To facilitate real-time monitoring of infection, we used a Staphylococcus aureus isolate that was made bioluminescent by inserting a modified lux operon into the bacterial chromosome. This bioluminescent reporter bacterium was used to study the antimicrobial effects of several antibiotics belonging to different molecular families. Treatment with rifampin, tobramycin, and ciprofloxacin was started 7 days after subcutaneous implantation of catheters precolonized with 10(4) CFU of S. aureus. Three different doses of antibiotics were administered twice a day for 4 consecutive days. The number of metabolically active bacteria in untreated mice and the tobramycin- and ciprofloxacin-treated groups remained relatively unchanged over the 4-week observation period, indicating poor efficacies for tobramycin and ciprofloxacin. A rapid dose-dependent decline in metabolic activity in rifampin-treated groups was observed, with almost a 90% reduction after two doses and nearly undetectable levels after three doses. The disappearance of light emission correlated with colony counts. After the final treatment, cell numbers rebounded as a function of concentration in a time-dependent manner. The staphylococci isolated from the catheters of mice treated with rifampin were uniformly resistant to rifampin but retained their in vitro susceptibilities to tobramycin and ciprofloxacin. Since the metabolic activities of viable cells and a postantibiotic effect could be detected directly on the support matrix nondestructively and noninvasively, the methodology is specifically appealing for investigating the effects of antibiotics on biofilms in vivo. Moreover, our study points to the possible use of biophotonic imaging for the detection of the development of resistance to therapeutic agents during treatment of chronic infections in vivo.

Journal Article

Animals, B16-F10-luc2, B16F10-luc2; Antibodies, Monoclonal/diagnostic use/immunology; *Diagnostic Imaging; Female; Fluorodeoxyglucose F18/diagnostic use; Glycoproteins/*immunology; Humans; Inflammation/*complications/immunology/pathology; Iodine Radioisotopes/diagnostic use/pharmacokinetics; Luminescent Measurements; Lymph Nodes/immunology/pathology/*radionuclide imaging; *Lymphangiogenesis; Lymphatic Metastasis; Melanoma, Experimental/*complications/immunology/pathology; Mice; Mice, Inbred C57BL; Mice, Transgenic; *Positron-Emission Tomography; Prognosis; Radiopharmaceuticals/diagnostic use; Skin/metabolism; Tissue Distribution; Vascular Endothelial Growth Factor C/metabolism; Vascular Endothelial Growth Factor Receptor-3/immunology

Metastasis to regional lymph nodes (LN) is a prognostic indicator for cancer progression. There is a great demand for sensitive and noninvasive methods to detect metastasis to LNs. Whereas conventional in vivo imaging approaches have focused on the detection of cancer cells, lymphangiogenesis within tumor-draining LNs might be the earliest sign of metastasis. In mouse models of LN lymphangiogenesis, we found that systemically injected antibodies to lymphatic epitopes accumulated in the lymphatic vasculature in tissues and LNs. Using a (124)I-labeled antibody against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we imaged, for the first time, inflammation- and tumor-draining LNs with expanded lymphatic networks in vivo by positron emission tomography (PET). Anti-LYVE-1 immuno-PET enabled visualization of lymphatic vessel expansion in LNs bearing metastases that were not detected by [(18)F]fluorodeoxyglucose-PET, which is clinically applied to detect cancer metastases. Immuno-PET with lymphatic-specific antibodies may open up new avenues for the early detection of metastasis, and the images obtained might be used as biomarkers for the progression of diseases associated with lymphangiogenesis.

Journal Article

Animals, B16-F10-luc2, B16F10-luc2; Base Sequence; Cell Line, Tumor; Cholesterol/metabolism; Ethylenediamines/*chemistry; Fibrosarcoma/metabolism/pathology; Gene Silencing; Humans; Injections, Intravenous; Liposomes/administration & dosage/chemical; synthesis/*chemistry/pharmacokinetics; Male; Mice; Mice, Inbred BALB C; Molecular Imaging; Phosphoric Acid Esters/*chemistry; Polyethylene Glycols/chemistry; RNA, Small Interfering/genetics/*metabolism; Spectrophotometry, Infrared

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.