reduced the number of candidates to 13, (including 4 of the original identified in both tissues and 9 identified in peritoneal only). The top 10 most mesothelial specific of these were then chosen for further screening by in-situ hybridization. In-situ hybridization of 10 candidate biomarkers Of the 10 candidate biomarkers identified in the study, several have previously been associated with the mesothelial lineage or mesothelioma, these include the prototype mesothelial lineage marker mesothelin (Msln)[15] and others such as uroplakin 3B(Upk3b) [35], prostaglandin I2 (prostacyclin) synthase (Ptgis) [39] and insulin-like growth factor binding protein 6 (Igfbp6) [40]. The remaining six, leucine rich repeat neuronal 4(Lrrn4), Na+/ K+ transporting ATPase interacting 4(Nkain4), proteoglycan 4(Prg4), matrix metallopeptidase 23(Mmp23), family with se- quence similarity 180, member A (Fam180a), selectin P(Selp) have not been previously associated with the mesothelial lineage. Although Prg4 specifically has not been previously associated with the mesothelial lineage, these cells are known to produce proteoglycans to facilitate lubrication within the peritoneum [41]. In-situ hybridization was carried out across a selection of mouse tissues including lung and heart (both with mesothelium intact), liver, kidney, spleen, intestine, testis and thymus. For the previously reported markers Msln, Upk3b, Ptgis and Igfbp6 mesothelial staining was observed. In addition two of the novel markers, Nkain4 and Lrrn4 showed mesothelial staining (Figure 1, Figure S1). LRRN4 is a novel human mesothelial lineage marker To confirm the specificity of these markers in human mesothelium and mesothelioma we designed primers against human MSLN, UPK3B, NKAIN4 and LRRN4 (Table S1) and tested their specificity by qRTPCR against a panel of 45 human Figure 1. In-situ hybridization images of mouse lung tissue with pleural mesothelium intact stained for A) mesothelin, B) uroplakin 3b, C) leucine rich repeat neuronal 4 and D) Na+/K+ transporting ATPase interacting 4. Note the mesothelial staining in blue. doi:10.1371/journal.pone.0025391.g001 LRRN4 and UPK3B as Mesothelial Markers total RNAs. The panel was selected to contain normal lung tissue, primary cells of the lung including a range of epithelial cells, smooth muscle cells and fibroblasts, primary mesothelial cells from 2 donors, 16 mesothelioma cell lines, 10 lung cancer cell lines and 8 unrelated cancer cell lines. Primary retinal pigment epithelial cells and urothelial cells were also included as unrelated epithelial cells. First considering the primary cells, MSLN, LRRN4 and UPK3B were detected in the primary mesothelial cells, however LRRN4 showed the greatest specificity and was only detected in primary mesothelial cells. MSLN and UPK3B however were less specific and were detected in bronchial epithelial cells and alveolar epithelial cells or retinal pigment epithelial cells and urothelial cells, respectively (Fig. 2). Despite this, testing the cell line panel revealed that MSLN still appears to be a better marker of mesothelioma than LRRN4 and UPK3B. MSLN was detected in 15 of the 16 mesothelioma lines tested and for 13 of these the expression level was higher than that of the primary mesothelial cells. In the case of LRRN4 and UPK3B the expression was only detected in 8 or 6 of the tested lines suggesting these markers are down-regulated or lost in cancer. Despite the higher fraction of mesothelioma lines positive for MSLN, it does not appear to be a completely mesothelioma specific marker as it was strongly expressed in the large cell lung carcinoma cell line T3M-10 and moderately expressed in the lung adenocarcinoma line PC-14. Surprisingly MSLN was also detected at high levels in the duodenal papillotubular adenocar- cinoma cell line TGBC18TKB and at moderate levels in the gastric signet ring carcinoma line Kato III, bladder transitional cell carcinoma line JMSU1, gall bladder carcinoma line TGBC2TKB and even weakly in the ovarian epitheloid line HeLa (Fig. 2). Finally at this stage of the screening NKAIN4 failed to validate as a marker of human mesothelium or mesothelioma, it was most highly expressed in primary urothelial cells and amongst the lung associated cell types it was much more highly expressed in alveolar and small airway epithelial cells than mesothelial cells (Fig. 2). Immunostaining of mesothelial biomarkers in human mesothelioma With the aim of developing these biomarkers for diagnostic testing in the future we set out to generate antibodies against UPK3B, and LRRN4. Three synthetic peptides were generated for each of the three proteins and antibodies raised in Rabbits (Figure S2). The best antibody for each protein was then chosen for further testing. Immunohistochemistry staining of a human epithelial mesothelioma sample was then carried out using these antibodies and a positive control against MSLN [29,30]. Mesothelial specific signal was observed for all three antibodies, with a pattern similar to that of the MSLN positive control (Fig 3). Cell surface staining was observed for MSLN, LRRN4 and UPK3B. Discussion We have described a comparative functional genomics screen to identify novel markers of the mesothelial lineage with potential for secretion. From this screen we identified several putative biomarkers and went on to generate antibodies against LRRN4, and UPK3B. In terms of specificity, LRRN4 appears to be the most specific marker of the mesothelial lineage, however when tested across a panel of 16 mesothelioma cell lines is only detected in 8, and down-regulated in the remaining 8 suggesting it may be lost during tumor progression (or adaptation to cell culture). We show that Mesothelin is detected in 15 of 16 cell lines and is upregulated relative to primary mesothelial cells in 13. PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25391