Abstract
BackgroundProtease activated receptor (PAR)-1 expression is increased in a variety of tumor cells. In preclinical models, tumor cell PAR-1 appeared to be involved in the regulation of lung tumor growth and metastasis; however the role of PAR-1 in the lung tumor microenvironment, which is emerging as a key compartment in driving cancer progression, remained to be explored.MethodsIn the present study, PAR-1 gene expression was determined in lung tissue from patients with non-small-cell lung cancer (NSCLC) using a combination of publicly available RNA microarray datasets and in house-made tissue microarrays including tumor biopsies of 94 patients with NSCLC (40 cases of adenocarcinoma, 42 cases of squamous cell carcinoma and 12 cases of other type of NSCLC at different stages).ResultsPAR-1 gene expression strongly correlated with tumor stromal markers (i.e. macrophage, endothelial cells and (myo) fibroblast markers) but not with epithelial cell markers. Immunohistochemical analysis confirmed the presence of PAR-1 in the tumor stroma and showed that PAR-1 expression was significantly upregulated in malignant tissue compared with normal lung tissue. The overexpression of PAR-1 in tumor stroma of NSCLC appeared to be independent from tumor type, tumor stage, histopathological differentiation status, disease progression and patient survival.ConclusionOverall, our data provide evidence that PAR-1 in NSCLC is mainly expressed on cells that constitute the pulmonary tumor microenvironment, including vascular endothelial cells, macrophages and stromal fibroblasts.
Highlights
Protease activated receptor (PAR)-1 expression is increased in a variety of tumor cells
Protease activated receptor-1 (PAR-1) gene expression is correlated with lung tumor stroma activation To explore the association of PAR-1 expression with the Non-small-cell lung cancer (NSCLC) stroma, we correlated PAR-1 gene expression levels with specific markers of different stromal cell types, including macrophages, endothelial cells, epithelial cells and fibroblasts in resected tumor specimens using publicly available microarray datasets
The commonly used differentiation marker for fibroblasts ACTA2 and markers for prominent constituents of extracellular matrix (ECM) deposition Collagen, type I, alpha (COL1A1) and Fibronectin (FN1) were all correlated with PAR-1 gene expression in the NSCLC specimens
Summary
Patients Tissue microarrays (TMAs, triplicate cores per case) were prepared with tumor sections obtained from NSCLC patients during surgery according to the guidelines of the Medical Ethical Committee of the Academic Medical Center of Amsterdam. The TMAs consist of samples from 94 patients with NSCLC, including 40 cases of adenocarcinoma (ADC), 42 cases of squamous cell carcinoma (SCC) and 12 cases of other type of NSCLC at different stages (Table 1). Mining of publically available RNA microarray dataset The datasets were derived from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/gds) using the R2 microarray analysis and visualization platform (http://r2.amc.nl). Correlation of gene expression between PAR-1 and markers of different stromal cell types in NSCLC cancer patients were derived by the R2 program from five different datasets, including Bild (n = 114, GSE3141), Peitsch (n = 150, GSE43580), EXPO (n = 121, GSE2109), Mao (n = 124, GSE 31852) and Hou (n = 156, GSE 19188). PAR-1 staining was performed with a primary antibody specific for PAR-1 (ATAP-2 ;1:200; SC13503, 24 h at 4 °C, Santa Cruz, San Diego, CA) [19, 20]. Results are expressed as mean ± SEM, P values < 0.05 are considered significant
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