Finally, E-cadherin expression could contribute to the migration mechanics used by the invading tumor cells. Gliomas are known for their diffusely infiltrative invasion pattern, in which the tumor cells migrate as single cells. However the heterogeneity of glioma genotypes, phenotypes, and surrounding tissue architecture suggest that a diversity of migration mechanisms could be used, even within the same tumor, at different stages of tumor dispersal. Although E-cadherin expression in gliomas is rare, when it occurs it could facilitate collective tumor cell migration strategies, including migration as discrete cell clusters or as multicellular strands or sheets, which are known to be dependent on cadherin expression [55]. One question is whether the pro-tumorigenic function of E- cadherin applies widely in gliomas or narrowly to GBM tumors with pseudo-epithelial differentiation. Arguing for the latter, ectopic overexpression of E-cadherin in the U87 glioma cell line did not increase, but rather slightly decreased migration in vitro (Lewis-Tuffin, unpublished observations). Unlike SF767 cells, which only express E-cadherin, U87 cells also express endogenous N-cadherin and cadherin-11, which could also affect migration- and proliferation-related signaling. Therefore predicting the effect of ectopic E-cadherin in GBM cell culture is not straightforward. This will be an important issue to explore in future studies. In conclusion, we have identified an unexpected association of E-cadherin expression with aggressive biologic behavior in a rare subset of GBM. The association of E-cadherin expression with adverse clinical behavior should be interpreted with caution, given the retrospective nature of the clinical/therapeutic data collected. Nonetheless, both our clinical and pre-clinical data support the conclusion that E-cadherin expression is associated with increased aggressiveness in human GBM. These findings provide a glimpse into the importance of the cadherin class of cell-cell adhesion molecules in the biology of high grade astrocytic tumors. Future studies should functionally dissect the specific molecular mecha- nisms involving cadherins and associated proteins in glioma biology. This will facilitate the identification of more reliable prognostic biomarkers, and perhaps the development of novel therapies. Finally, our findings may also be relevant to epithelial tumor biology. Despite the significance of EMT in epithelial tumor progression and dissemination, many epithelial metastases retain E-cadherin expression. The possibility that deregulated E- cadherin function may promote the aggressiveness of these tumors warrants further investigation. Materials and Methods Ethics Statement All patient-related studies were approved by the Mayo Clinic Institutional Review Board and therefore have been performed in accordance with the ethical standards laid down in the Declaration of Helsinki, including obtaining written informed consent prior to donation of tissue. Animal studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC A5409) and were conducted according to Mayo Clinic IACUC guidelines for animal husbandry. GBM tissue microarray tumor specimens and immunohistochemical analysis Construction of the two Mayo Clinic human tissue microarrays was performed as described in a previous publication [33]. Briefly, 83 cases of human high grade astrocytomas (11 cases of grade III astrocytomas, 72 cases of GBMs) were used to construct two tissue microarrays consisting of 3–4 0.6 cm diameter cores per tumor. E-Cadherin Expression in GBM Each tissue microarray also contained samples of non-neoplastic gray and white brain matter (obtained from epileptigenic brain), infiltrating ductal carcinoma breast tissue, six GBM xenograft cell lines, and U87 and U251 conventional glioma cell lines. The human glioma rim-core tissue microarray was a gift from Michael E. Berens (Translational Genomics Research Institute, Phoenix, AZ) and has been described previously [34]. Immunohistochem- istry for E-cadherin was performed on the three tissue microarrays as described previously [33] using a monoclonal human anti E- cadherin antibody (clone 4A2C7, Zymed/Invitrogen, Carlsbad, CA). Tumor specimens and immunohistochemical analyses of GBMs with epithelial/pseudo-epithelial differentiation Tumor classifications, tissue microarray construction, immuno- histochemistry to analyze E-cadherin expression, and immunohis- tochemical scoring were described in detail in a previous publication [33]. Briefly: tumors from 32 patients from a previously characterized retrospective series of glioblastomas with varying degrees of epithelial morphology were stained with a monoclonal human anti E-cadherin antibody (clone 4A2C7, Zymed/Invitrogen, Carlsbad, CA) using tissue microarray sec- tions. These tumors represent a rare subtype of GBM (,2%) and were largely derived from pathology consultations. Clinical follow- up was necessarily limited, but present in 27 patients (84%): 7 women and 20 men. The median age at diagnosis was 56 years (interquartile range 44–67). The tumors were histologically classified by previously published criteria as adenoid-GBM (n = 11), epithelioid GBM (n = 4) or GBM with true epithelial differentiation (n = 12). Postoperative treatment approaches in- cluded radiation therapy to the brain (50–60 Gy)(n = 15), with chemotherapy (n = 6), including Temodar in 5 patients, or precise treatment modality unknown (n = 12). Immunohistochemical scoring as either positive or negative was performed by a single, blinded neuropathologist (F.J.R.). Univariate analyses of E- cadherin immunoexpression with respect to overall and recur- rence-free survival, as well as relevant clinicopathologic features were performed using the log-rank test, and illustrated with Kaplan Meier curves. All analyses were 2-sided with p-values ,0.05 considered statistically significant. Xenograft information Each of the 19 serially passaged xenograft cell lines used in this study are derived from resected tumors from different human patients and are propagated by serial transplantation in the flank of nude mice [38]. Tumors are propagated exclusively in vivo in order to preserve molecular and histopathologic features of the primary patient tumor specimens. Eighteen of the xenograft cell lines have been described previously [38,56–58]. GBM59 has not been reported previously; the original tumor from which GBM59 was derived was also diagnosed as a GBM (Carlson and Sarkaria, unpublished manuscript). Orthotopic xenograft model and evalution of relative invasiveness Short-term explant cultures derived from flank tumor xeno- grafts were injected orthotopically into nude mouse brain as described previously [58]. Mice were euthanized when they reached a moribund condition. Brains from these mice were resected and then bisected along the needle tract used for injecting the tumor cells. Half of each bisected brain was placed in formalin and subsequently embedded in paraffin. These tissue samples were processed for routine H&E staining in the TACMA Mayo core PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13665