developmental example of EMT underlies the formation of the neural tube, allowing N-cadherin expressing cells in the developing neuroepithelium to separate and migrate away from surrounding E-cadherin expressing cells [14,15]. Further rounds of differential cadherin expression are also thought to promote regionalization during later stages of nervous system development [16,17]. The classical cadherin superfamily (both Types I and II) consists of more than 18 members, the differential expression of which occurs at all stages of nervous system development and persists into adulthood. Neurons that express a particular cadherin preferen- tially form synapses with, and migrate into regions that express the same cadherin [17,18]. Thus differential cadherin expression is a general mechanism for regulating cell motility and promoting brain regionalization. During epithelial tumor progression this normal morphogenetic process is hijacked, and EMT is thought to induce the invasion and metastasis of, among others, breast, kidney, and colorectal cancer cells [6,15,19]. Previous examinations of cadherin expression in the normal human adult nervous system indicate that E-cadherin expression is rare, and limited to the arachnoid membrane [20–25]. In mice, E- cadherin is also expressed in neural stem cells, where it regulates self renewal [26]. On the other hand, mesenchymal cadherins such as N-cadherin or cadherin 11 are expressed in multiple brain regions, including the cortex and hippocampus, and are important to normal nervous system function [18], with a possible role in gliomagenesis or invasiveness. Only a handful of studies have focused on the involvement of cadherins or catenins in high-grade adult gliomas (anaplastic astrocytomas or GBMs) [22–25,27–29] (reviewed in [30]). Most of these studies examined resected tumors or glioblastoma cell lines and focused on the expression of E- cadherin and N-cadherin protein. Consistently, E-cadherin protein expression was found to be rare to non-existent in both tumor and normal brain tissue [20,22,24,25,31], while N-cadherin was extensively represented. In contrast to these results, two recent studies argue that E-cadherin expression decreases with brain tumor grade when compared to normal brain [27,32], raising the possibility that a classic EMT is involved in glioma progression. On balance, the existing data are inconclusive as to the pathobiologic role of E-cadherin in high-grade adult gliomas. We show here that E-cadherin expression is very limited in normal brain, and rare in astrocytic and GBM tumors. Previously we had examined the histopathologic, immunophenotypic, and molecular characteristics of a rare form of GBM exhibiting varying degrees of epithelial or pseudo-epithelial differentiation [33]. In this study we found E-cadherin protein to be expressed in one third of these tumors, and this expression correlated with worse patient outcome. Therefore we sought to determine if the expression of E-cadherin had functional consequences related to tumor biology. Using multiple cell line models of GBM, it was determined that expression of E-cadherin correlated with growth, migration, and invasiveness. Taken together, our data suggest a novel and potentially important role for E-cadherin in the lethality of this subset of high-grade adult gliomas. Results E-cadherin expression is rare in GBM We used immunohistochemistry to examine E-cadherin expres- sion in brain tumor and non-neoplastic brain samples from three tissue microarrays (TMAs): two constructed at the Mayo Clinic containing a total of 83 glioblastoma (predominantly) or anaplastic astrocytoma samples and one glioma invasion tissue microarray containing 31 matched brain tumor rim/core samples (a generous gift from M. Berens [34]). We initially probed the TMAs with the E-Cadherin Expression in GBM clone 36 E-cadherin antibody, which is highly specific for E- cadherin when used with Western Blot. However, in the context of the formalin-fixed, paraffin-embedded samples of the TMAs, this antibody produced non-specific staining (Figure S1), similar to what has been reported previously [35]. Therefore the 4A2C7 antibody clone, which does not have this problem (Figure S1) was used to evaluate E-cadherin expression. While E-cadherin staining was clearly present in the infiltrating ductal breast carcinoma samples on these TMAs, we did not find any E-cadherin expression in any brain tumor or non-neoplastic brain sample (data not shown). Thus brain- and brain tumor-associated E- cadherin expression is rare. E-cadherin expression in GBM with epithelial/ pseudoepithelial differentiation We also performed immunohistochemistry to analyze E- cadherin protein expression in tissue samples from 27 individual cases with adequate clinical follow up of a rare sub-type of GBM with epithelial/pseudoepithelial differentiation. These tumors likely represent primary GBM based on a relatively short duration of symptoms in patients with available history and lack of a documented lower grade precursor per established criteria [36]. In addition, molecular genetic changes typical of primary GBM were present, including EGFR amplification (25%) or gain of chromo- some 7 without amplification (50%), and whole chromosome 10 loss (57%). E-cadherin expression was present in 9 cases (33%). The majority of the E-cadherin positive cases (8 out of 9 cases) exhibited a restricted pattern of E-cadherin immunostaining, in which E-cadherin was focally expressed in discrete nests of tumor cells, usually reflecting areas of epithelial-like differentiation (Figure 1A, Ai). In these positive cells, E-cadherin was expressed primarily on the plasma membrane, but was dispersed, rather than concentrated at areas of homotypic cell-cell contact as would be expected (see [37] for an example of normal E-cadherin distribution). In a single case (Figure 1B, Bi) E-cadherin expression was more diffusely spread throughout most cells in the epithelial component of the tumor. In this tumor, E-cadherin immunostain- ing was both membranous and cytoplasmic. In contrast to E- cadherin expression, b-catenin was present in all cases studied (Figure 1C), with a membranous and cytoplasmic (56%) or membranous only (44%) staining pattern. Patients whose tumors expressed E-cadherin demonstrated poorer overall survival compared to those that did not (Figure 1D; statistically significant at p = 0.021, log rank test). Although E-cadherin immunostaining was more frequent in GBM with epithelial-like differentiation (58%) compared with the adenoid (A-GBM) and epithelioid (E- GBM) histologic subgroups (13%) [33], only E-cadherin expres- sion was associated with survival. In contrast, there were no significant associations between survival and age, tumor location, tumor size, extent of resection, b-catenin immunostaining, molecular cytogenetic abnormalities, or proliferative indices, nor between E-cadherin immunostaining and any of those parameters in this patient population (p.0.05). E-cadherin expression in the GBM xenograft model Because the expression of E-cadherin in normal central nervous system tissue is rare and correlated with worse outcome for patients with GBM, we used an experimental model of GBM to follow up this observation. The Mayo GBM xenograft model is comprised of 19 primary GBM xenografts that were established directly from patient tumor specimens and passaged exclusively in vivo as flank xenografts. Orthotopic implantation of short-term explant cultures derived from these xenograft lines results in tumors with morphologic and genetic features similar to those seen in the PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13665