LRRN4 and UPK3B as Mesothelial Markers Figure 2. Expression of A) MSLN, B) LRRN4, C) UPK3B, D) NKAIN4 across a panel of primary human cell types and cancer cell lines measured by qRTPCR. doi:10.1371/journal.pone.0025391.g002 Interestingly qRTPCR across a panel of human RNAs demonstrated that MSLN is not restricted to mesothelium or mesothelioma. In normal primary lung associated cells MLSN is detected at a higher level in bronchial epithelial cells than mesothelial cells. In tumor cell lines, expression was detected in mesothelioma cell lines, but was also expressed at similar levels in a large cell lung carcinoma, duodenal papillotubular adenocarcinoma, signet ring carcinoma, transitional cell carcinoma and gall bladder carcinoma. Weak expression in HeLa cells was also observed, which is consistent with a list of biomarkers reported as upregulated in cervical cancer [42]. MSLN is also upregulated in other cancers including ovarian cancer, and pancreatic ductal adenocarcinoma [43] and in its original description, soluble mesothelin was found to be a serum marker for ovarian cancer [17]. This has implications for the MESOMARK [12,13] kit for detection of soluble mesothelin as other tumor types including some lung cancers with similar presentations may produce soluble mesothelin. Regarding the other two markers. UPK3B has been reported previously as a marker of the mesothelial lineage [35]. However as its name suggests it is also expressed in urothelial cells and we also find strong expression in retinal pigment epithelial cells. LRRN4 however has not been previously associated with mesothelioma or the mesothelial lineage. LRRN4 (also known as NLRR4 and C20orf75) is a leucine rich repeat protein that was originally identified as a neuronal protein involved in long term memory formation [44], interestingly in this publication they note strong expression in the lung and ovary, similar to the pattern for mesothelin. Although LRRN4 has not been associated with mesothelioma it has been shown previously to be regulated in response to acute lung injury [45] and in Chronic Obstructive Pulmonary Disease (COPD) [46] suggesting it may play a role in response to lung damage. It is also upregulated in the adipose of Mif knockout mice [47]. MIF (macrophage migration inhibitory factor) is implicated in COPD [48] and is a cytokine involved in maintenance of proinflammatory response in macrophages [49]. It is hypothesized that there is a link between asbestos exposure, chronic inflammation and mesothelioma [50] however whether there is any significance in this link is unclear. The observation that LRRN4 is highly expressed in the primary mesothelial cells, is lost in 8 of the mesothelioma cell lines and is Figure 3. Immunohistochemisty of a human epithelial mesothelioma stained with antibodies against MSLN, LRRN4, UPK3B. doi:10.1371/journal.pone.0025391.g003 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25391