Project description:Introduction: Pleural mesothelioma (PM) is known as one of the most aggressive malignancies among all cancers, despite of usually absent tumor-driver mutations in oncogenes. It develops in a unique inflammatory tumor microenvironment (TME), which has been postulated as major contributor of PM’s highly aggressive nature. Mesothelioma-associated fibroblasts (Meso-CAFs), a main component of the TME have recently been shown to substantially stimulate several aspects of PM aggressiveness and promote the malignant transformation of pleural mesothelial cells. However, respective cell models for TME research in PM are still very limited. The most commonly used pleural mesothelial cell line Met5A has been established decades ago and patient-derived Meso-CAFs have just recently been characterized for the first time. The aim of the current study was to generate and characterize pleural mesothelial and Meso-CAF cell models with an extended life span that closely resemble the primary cells isolated from human tissue. Methods: Pleural mesothelial cells and Meso-CAFs were isolated from human tissue of pneumothorax and PM patients, respectively. Retroviral transduction was used to induce stable expression of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) in the primary cells. The established cell models were evaluated by measuring their doubling times, gene expression and protein activity levels of hTERT, as well as the absolute lengths of telomeres. The transduced cells were compared to their primary counterparts on the protein level using proteome analysis and the impact of their conditioned media (CM) on tumor cell growth was investigated by videomicroscopy. Results: All transduced derivatives exhibit elevated hTERT gene expression and protein activity, increased hTERT protein amounts in the nucleus, and moderately higher absolute telomere lengths compared to their parental primary cells. The transduction with hTERT did not elicit marked changes in the morphology of the cells, as well as in their proteomes and secretomes. The CM of primary and hTERT-transduced Meso-CAFs comparably stimulated PM cell growth, while medium conditioned by normal pleural mesothelial cells including their hTERT-transduced derivatives was not able to induce a growth stimulating effect. Conclusion: The hTERT-transduced cells closely resemble their primary counterparts, while retaining telomerase activity, thus preventing replicative senescence. The new cell models provide valuable tools for the investigation of cellular interactions cellular interactions within the TME of PM and thus may help to identify novel biomarkers for early diagnosis and to develop new therapeutic strategies.

Project description:Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n=40), normal lung specimens (n=4), normal pleura specimens (n=5), and MPM and SV40-immortalized mesothelial cell lines (n=5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma which correlate loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (i.e. software) to create and explore complex physiological pathways that may be relevant in mesothelioma tumorigenesis, pathobiology, or both. Tissues and cell lines profiled using microarrays. Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Bostonâs Brigham and Womenâs Hospital (BWH) between October 1998 and August 2000. All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed. All normal specimens were obtained from patients who were never diagnosed with MPM. Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH. The JMN1B MPM cell line19,20 has been described previously. The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection. Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM. All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used). The microscopic slides from the patientsâ resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases. Linked clinical and pathological data were obtained for all patients who contributed tumor specimens. Specimens and data were rendered anonymous to protect patient confidentiality. Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH. SUBMITTER_CITATION: Gordon, G. J., Rockwell, G. N., Jensen, R. V., Rheinwald, J. G., Glickman, J.N., Aronson, J. P., Pottorf, B. J., Nitz, M. D., Richards, W. G., Sugarbaker, D. J., and Bueno, R. Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling. American Journal of Pathology, 166: 1827-1840, 2005.

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Contribution of crosstalk of mesothelial and tumoral epithelial cells in pleural metastasis of lung cancer

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