Hypoxia increases membrane metallo-endopeptidase expression in a novel lung cancer ex vivo model - role of tumor stroma cells

Background: Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo.Methods: Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays.Results: Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase (MME, neprilysin, CD10) expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets.Conclusions: The novel ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced aggressiveness of hypoxic cancers

A GAMMA-RAY ATTENUATION METHOD FOR VOID FRACTION DETERMINATIONS IN EXPERIMENTAL BOILING HEAT TRANSFER TEST FACILITIES

Gamma rays emanating from a radioactive source are beamed through and are attenuated by steam --water mixtures contained in a simulated reactor flow channel. The emergent radiation is detected by a scintillation crystalphotomultiplier tube assembly. An expression is developed which yields the void fraction when the detector output with the channel empty, filled with water, and containing the mixture in question is known. The principal sources of errors inherent in the method are analyzed and their magnitudes computed for a specific test facility. With a uniform bubble distribution on this facility, the maximum possible error in void fraction is approximately plus or minus 0.03. The method is also applied to three idealized preferential phase distributions simulated in Lucite. However, the large discrepancy between calculated and measured void fractions illustrates the need for more refined experimental techniques where non-uniform distribution of voids are encountered. Such techniques are being developed. (auth

Causes of pulmonary granulomas: A retrospective study of 500 cases from seven countries

PubMedID: 22011444Background: The frequencies of various causes of pulmonary granulomas in pathological material are unknown, as is the influence of geographical location on aetiology. The aim of this study was to identify the causes of pulmonary granulomas in pathological specimens, to define their frequencies, and to determine whether these causes vary by geographical location. Methods: 500 lung biopsies and resections containing granulomas were reviewed retrospectively by expert pulmonary pathologists from 10 institutions in seven countries. Fifty consecutive cases from each location were assigned a diagnosis based on histological features and available clinical/microbiological data. Results: A specific cause was identified in 58% of cases (290/500), most commonly sarcoidosis (136, 27%) and mycobacterial or fungal infections (125, 25%). Mycobacteria were identified in 19% of cases outside the USA versus 8% within the USA. In contrast, fungi accounted for 19% cases in the USA versus 4% in other locations. Fungi were mostly detected by histology, whereas most mycobacteria were identified in cultures. In 42% of cases (210/500) an aetiology could not be determined. Conclusions: Across several geographical settings, sarcoidosis and infections are the most common causes of pulmonary granulomas diagnosed in pathological specimens. Fungi are more commonly identified than mycobacteria in the USA, whereas the reverse is true in other countries. A definite aetiology cannot be demonstrated in more than a third of all cases of pulmonary granulomas, even after histological examination. These findings highlight the need to submit material for histology as well as cultures in all cases in which granulomatous disease enters the differential diagnosis

Impacts: planning a European initiative for standardized protein analysis of archival clinical tissues

Background The lack of standardized guidelines for protein analysis of archival tissues (AT) seems to have hampered the use of such samples in molecular analyses. IMPACTS is a new European consortium aiming to establish guidelines for AT analyses. Here we focus on the evaluation of protein analysis in AT, fixed with formalin or new fixatives, for research and diagnostic use. Methods Tissue samples were fixed with formalin (FFPE) or a new fixative (FineFix, Milestone Srl) and inspected by histology. Five sections from three matched samples of each tissue were distributed within the consortium in order to compare the efficiency of protein recovery by immunohistochemistry, Western blot, and protein lysate microarray technology. Results By applying an optimized protocol, proteins can be successfully extracted from FFPE and FineFix tissues. Moreover, we could show a correlation between immunohistochemical, Western blot, and protein lysate microarray results for each of the tissue fixation procedures. Differences between methods, however, were observed in the total protein yield and staining intensities for antibodies detecting membrane proteins, including E-cadherin, EGFR, and HER2. Conclusion(s) Analysing proteins from fixed tissue samples can provide critical information for clinical decision making. Although long-term studies for formalinsubstitutes such as FineFix are still missing, formalin-free tissue fixation represents a reliable alternative for protein preservation, regarding quality and quantity, as compared to FFPE tissue. The IMPACTS consortium is working towards evaluating AT sample processing for protein analysis and tap into the vast treasure chest of archived samples