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

Relative abundance of fungal microbiota in lower respiratory tract samples.

<p>Healthy adults (bar group 1a), patients with healthy respiratory tract but with antibiotic therapy for extrapulmonary infection (bar group 1b), non-neutropenic intubated and mechanically ventilated ICU patients without antibiotic therapy (bar group 2a), non-neutropenic intubated and mechanically ventilated ICU patients with antibiotic therapy for extrapulmonary infection (bar group 2b), and patients with antibiotic therapy due to pneumonia (bar group 3b). Fungi are shown at genus level. Yellow bars represent the sum of <i>Candida</i> sequences across all samples per group related to the total number of detected fungal sequences within each of the groups. By conventional culture <i>Candida spp</i>. were detected in 0/8 (group 1a), 0/7 (group 1b), 1/7 (group 2a), 3/6 (group 2b), and 11/26 (group 3b) patients. No other fungi were cultured.</p

Demographic data and clinical characteristics of study patients.

<p>ICU patients were all intubated and mechanically ventilated.</p

<i>Candida</i> positive cultures from the oral cavity, lower respiratory tract (LRT), and the <i>Candida</i> colonization index.

<p><i>Candida</i> positive cultures from the oral cavity, lower respiratory tract (LRT), and the <i>Candida</i> colonization index.</p

Relative abundance of bacterial microbiota in consecutively sampled patients.

<p>Consecutive lower respiratory tract samples of 4 intubated and mechanically ventilated patients with pneumonia. Bacteria are shown at genus level. Unclassified sequences in sample 304-3B-ASP-0 revealed <i>Escherichia/Shigella sp</i>. using a confidence threshold of 0.63. Comparison of these sequences with reference sequences using BLAST yielded uncultured <i>Shigella sp</i>. 16s rRNA genes. Unclassified sequences in sample 313-3B-VAP-0 revealed <i>Enterobacter sp</i>. using a confidence threshold of 0.63. Comparison of these sequences with reference sequences using BLAST yielded uncultured proteobacterium 16s rRNA genes. VAP = Ventilator associated pneumonia, ASP = aspiration pneumonia, NAP = nosocomial acquired pneumonia.</p

Bray-Curtis distances between study groups indicating differences in fungal species composition.

<p>Distance of zero indicates that groups are completely similar for every species. Distance of 1 indicates that groups are completely dissimilar and do not share any species. Group 1a, healthy adults; group 1b, healthy respiratory tract but with antibiotic therapy for extrapulmonary infection; group 2a, non-neutropenic intubated and mechanically ventilated ICU patients without antibiotic therapy; group 2b, non-neutropenic intubated and mechanically ventilated ICU patients with antibiotic therapy for extrapulmonary infection; group 3b, non-neutropenic intubated and mechanically ventilated ICU patients with antibiotic therapy due to pneumonia.</p

Average relative <i>Candida</i> abundance in lower respiratory tract samples.

<p>Healthy adults (bar group 1a), patients with healthy respiratory tract but with antibiotic therapy for extrapulmonary infection (bar group 1b), non-neutropenic intubated and mechanically ventilated ICU patients without antibiotic therapy (bar group 2a), non-neutropenic intubated and mechanically ventilated ICU patients with antibiotic therapy for extrapulmonary infection (bar group 2b), and patients with antibiotic therapy due to pneumonia (bar group 3b). Significant differences of average relative <i>Candida</i> abundance between groups are shown with bars representing the average <i>Candida</i> abundance and standard errors in given groups (* = p <0.05 for groups 2a, 2b, 3b versus 1a or 1b).</p