Citation Details

Journal Article

IVIS, Xen29, Xen 29, Staphylococcus aureus Xen29, Animals; Blood Glucose/metabolism; Cell Adhesion/drug effects; Cell Count; Cell Movement/drug effects; Chemokine CXCL2/pharmacology; Diabetes Mellitus, Type 1/blood/complications/*immunology/microbiology; Diabetes Mellitus, Type 2/blood/complications/*immunology/microbiology; Diet, High-Fat/adverse effects; Disease Models, Animal; Hyperglycemia/chemically induced/complications; Inflammation/blood/complications/immunology/microbiology; Leukocytes/cytology/drug effects/*immunology/microbiology; Male; Mice; Mice, Inbred C57BL; Phagocytes/cytology/drug effects/microbiology; Staphylococcus aureus/physiology

BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62+/-18% and 85+/-21%, respectively) and high fat diet-induced (77+/-25% and 86+/-17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3+/-1.0) compared to control (8.0+/-1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.

Journal Article

IVIS, Xenogen

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of beta-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (microCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.

Journal Article

MDA-MB-231-luc2, IVIS, Breast Cancer, Bioware, Angiogenesis Inhibitors/pharmacology; *Cell Communication/drug effects; Cell Proliferation/drug effects; Extracellular Matrix/drug effects/metabolism; Fibroblasts/drug effects/metabolism/pathology; Gene Silencing/drug effects; Human Umbilical Vein Endothelial Cells/drug effects/metabolism; Humans; Intercellular Signaling Peptides and Proteins/pharmacology; Luminescent Measurements; Matrix Metalloproteinase 14/metabolism; Microscopy, Fluorescence, Multiphoton; *Models, Biological; Neoplasms/*blood supply/enzymology/*pathology; Neovascularization, Pathologic/*pathology; Signal Transduction/drug effects; Spheroids, Cellular/drug effects/enzymology/pathology; Stromal Cells/drug effects/pathology; Tumor Cells, Cultured

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.

Journal Article

HCT-116-luc2, HCT116-luc2, IVIS, Animals; Antibodies, Monoclonal/administration & dosage/*immunology; Antigens, CD45/metabolism; Cell Line, Tumor; Cell Transformation, Neoplastic/pathology; Disease Models, Animal; Flow Cytometry; Fluorescent Dyes/*metabolism; Humans; Imaging, Three-Dimensional/*methods; Injections, Intravenous; Leukemia/*diagnosis/*pathology; Leukemia, Myeloid, Acute/pathology; Longevity; Luminescent Measurements; Mice; Mice, SCID; Reproducibility of Results; Spectroscopy, Near-Infrared/*methods

BACKGROUND: The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. METHODS: Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. RESULTS: The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. CONCLUSIONS: A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

Journal Article

A549-luc-C8, A549-luc, IVIS, Bioware

Nanoparticles (NPs) have emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before clinical application can occur. Here the use of gold nanoparticles (AuNPs) to detoxify the antitumoral agent cisplatin, linked to a nanoparticle via a pH-sensitive coordination bond for endosomal release, is presented. The NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and results in an absence of observed toxicity. Besides, AuNPs present unique opportunities as drug delivery scaffolds due to their size and surface tunability. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalized in cells and cisplatin is released. Additionally, the possibility to track the drug (Pt) and vehicle (Au) separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.