Proteomics-Based Identification of Proteins Secreted in Apical Surface Fluid of Squamous Metaplastic Human Tracheobronchial Epithelial Cells Cultured by Three-Dimensional Organotypic Air-Liquid Interface Method

Abstract

Squamous cell carcinoma in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared with normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified by liquid chromatographytandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins [SCC antigen 1 (SCCA1), SCC antigen 2 (SCCA2), S100A8, S100A9, Annexin I, and Annexin II] in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens. [Cancer Res 2007;67(14):6565-73]

Overexpression of squamous cell carcinoma antigen in idiopathic pulmonary fibrosis: clinicopathological correlations

“…Other works have recently described nuclear–cytoplasmic positive staining of serpin (maspin and SCCA) in non-small cell lung carcinoma. In this work, the nuclear maspin positivity was correlated with a better survival than with cytoplasmic staining 32. The synthesis of SCCA is thought to be essentially in the cytoplasm.…”

Growth Suppression of Lung Cancer Cells by Targeting Cyclic AMP Response Element-Binding Protein

“…Cells were cultured in RPMI containing 10% FBS. Normal human tracheobronchial epithelial (NHTBE) cells (Clonetics, San Diego, CA), which were used as the control, were cultured by a three-dimensional organotypic air-liquid interface method as described previously (31)(32)(33)(34)(35). Ro-31-8220 was purchased from Calbiochem (La Jolla, CA) and prepared in DMSO.…”

High-Throughput and Targeted in-Depth Mass Spectrometry-Based Approaches for Biofluid Profiling and Biomarker Discovery

“…Analysis and identification of proteins in secretomes using proteomics . Tissue SCC antigen 1 and 2 [105] Sardana et al Tissue Not reported in this study [108] Pardo Tissue serpins [110] Chevallet et al Serum, tissue Dihydrodiol dehydrogenase [98] Chen et al…”

“…This new source is being increasingly interrogated by the clinical proteomics community, as is Biomarkers Med. (2007) 1(4) future science group future science group apparent from the rapid rise in publications: a PubMed search using various combinations of the keywords 'secretome' or 'cell-conditioned medium' and 'proteomics' or 'mass spectrometry' yielded two primary papers in 2004 [88,89], four papers in 2005 [90][91][92][93], six papers in 2006 [94][95][96][97][98][99] and already ten papers mid-2007 [100][101][102][103][104][105][106][107][108][109][110]. Importantly, half of the studies (11) describe differential analyses between cancer cell types or stages and have validated selected secretome candidates in patient material using antibodybased assays in serum or tissue microarrays.…”

“…A combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 η had a higher sensitivity (95%) and specificity (77%) for cancer stage prediction than any single marker. Another recent notable paper, by Kim et al is the analysis of apical surface fluid of squamous metaplastic human tracheobronchial epithelial cells harvested from 3D organotypic cultures [105]. A 2D gel-based differential analysis was performed between aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells.…”