In vivo molecular microimaging of pulmonary aspergillosis.

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy.

Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo.

Mesothelial vitronectin stimulates migration of ovarian cancer cells.

Ovarian carcinomas, the most fatal gynaecological malignancies, are associated with poor prognosis predominantly because of a high recurrence rate. Ovarian cancer cells spread widely throughout the abdominal cavity leading to peritoneal metastasis. The influence of the mesothelial microenvironment on the biological mechanisms leading to cancer cell colonization of the mesothelium is poorly understood. This study aims to investigate whether mesothelial secretions affect the migration of ovarian cancer cells and focuses on the role of the adhesive molecule Vn (vitronectin) and its integrin receptors. An in vitro co-culture model indicated that clusters of IGROV1 and SKOV3 cells adhere to MeT-5A mesothelial cells preferentially at intercellular sites, invade the mesothelial monolayer and alter the integrity of the mesothelium. In addition, mesothelial CM (cell-conditioned medium) induces migration of IGROV1 and SKOV3 cells in Boyden chambers and wound healing assays. Furthermore, blocking molecules directed against vitronectin or its alphav integrin receptor decrease mesothelial-CM-induced migration by approximately 40% and 60-70% for IGROV1 and SKOV3 ovarian cancer cells, respectively, in Boyden chamber assays. Wound healing assays that allow cell migration to be measured over 24 h periods demonstrated that blocking molecules prevent the migration of IGROV1 and SKOV3 cells. Vitronectin is present in CM MeT-5A (mesothelial conditioned medium) and in metastatic peritoneal tissue sections. The expression of vitronectin at the periphery of mesothelial cells and within ovarian cancer cell clusters suggests a potential role for this molecule during intraperitoneal implantation of ovarian cancer cells. Vitronectin could represent a target for the development of anti-adhesive strategies to impede ovarian cancer dissemination.

Vitronectin and its receptors partly mediate adhesion of ovarian cancer cells to peritoneal mesothelium in vitro.

Epithelial ovarian cancer cells metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Adhesive molecules that lead to this implantation remain unclear. The aim of our study was to focus on the role of vitronectin (Vn) and its receptors, alpha(v) integrins and urokinase plasminogen activator receptor (uPAR), in the interactions of ovarian adenocarcinoma cells (IGROV1 and SKOV3 cell lines) with mesothelial cells (MeT-5A cell line and primary cultures). For all cell lines, immunofluorescence staining disclosed the presence of Vn over the whole cell surface and in thin continuous deposits underlining the cell periphery. Recruitment of Vn receptors to cell-cell contact sites was also revealed. We developed two distinct methods for the evaluation of in vitro cell-cell adhesion using cocultures of the tumor and mesothelial cells. Both adhesion assays revealed a strong ability of ovarian cancer cells to adhere preferentially to mesothelial intercellular junctions. Adhesion of ovarian carcinoma cells to mesothelial cells was significantly inhibited using anti-Vn-, -alpha(v)-integrin- and -uPAR-blocking antibodies or cyclic peptide cRGDfV. These results evidence the ability of ovarian carcinoma cells to bind to peritoneal mesothelium in vitro and strongly suggest that Vn and its receptors contribute to this crucial event.

Adhesion of human ovarian adenocarcinoma IGROV1 cells to endothelial cells is partly mediated by the alphav integrins-vitronectin adhesive system and induces an alteration of endothelial integrity.

Several lines of evidence suggest that vascularization plays an important role in the growth, local expansion and dissemination of ovarian epithelial tumours. However, the interaction of ovarian carcinoma cells with the endothelium remains poorly understood. To investigate adhesive events underlying this process, we used an in vitro model of cocultures between the IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVECs). IGROV1 cells were shown to adhere rapidly on the HUVECs monolayer. Adhesion was inhibited by anti-alphav integrin and anti-Vn blocking antibodies, but not by anti-beta1 integrin antibodies. Anchorage of carcinoma cells led to the rupture of endothelial integrity, as revealed by the formation of holes in the monolayer and by the disappearance of the interendothelial VE-Cadherin network. Considering the ability of ovarian carcinoma to disseminate by a haematogenous way, these in vitro events could mimic a preliminary step for carcinoma cells crossing the endothelial barrier to extravasate.