Abstract 3313: A proliferating population of glioma stem-like cells as characterized by expression of the cell surface glycoprotein CD58

Glioblastomas (GBMs) are among the most common and most lethal primary brain tumors, characterized by a high cellular proliferation and invasion into brain parenchyma. Glioma stem-like cells (GSCs) have been proposed as being responsible for tumor initiation and recurrence following conventional therapy with chemoradiation. Attempts to identify a single cell surface marker which specifically identifies GSCs within human tumors have had limited success. In an effort to refine GSC identification through the use of a panel of cell surface markers, we identified the glycoprotein CD58 as a marker which more selectively identifies GSCs when added to established markers, such as CD133, podoplanin (pdpn), or CD15. To characterize the frequency of CD58 expression in neurosphere forming primary culture cell lines and freshly obtained surgical specimens, cells were subjected to flow cytometry using an anti-CD58 monoclonal antibody. In vitro analysis of self-renewal, a principle feature of GSCs, was performed with the neurosphere assay. Treatment resistance was compared by radiating single cell suspensions with 2 Gy ionizing radiation prior to neurosphere formation. Cell cycle was measured by flow cytometry and S-phase by incorporation of EdU. Survival analysis was performed on archival tumor specimens following immunohistochemistry with an anti-CD58 monoclonal antibody. Using a panel of 14 GSC primary cultures, CD58 expression was found in all lines, with a median expression of 85.4% of cells, ranging from 8.7% to 100%. CD58 expression was found in 67% of fresh GBM specimens, with expression ranging from 5% to 89% of tumor cells (median 9.4). Culturing of cells beyond 5 passages led to further enrichment for CD58 expressing cells. Neurosphere assays demonstrated an increase in self-renewal in cells expressing CD58 compared to pdpn/CD133/CD15 alone. To investigate whether this difference was a result of increased proliferation of CD58 cells, we performed cell cycle analysis and found a significantly higher percentage of CD58 expressing cells in S-phase (mean of 23.2% vs. 7.9% for CD58 expressing vs. non-expressing, respectively). Immunofluorescence of archival specimens with MIB1/Ki-67 revealed preferential staining in CD58 expression cells. In cell mixing experiments, we observed that CD58 expressing cells rapidly outgrew CD58 non-expressing, highlighting the differences in proliferative capacity. Analysis of patients based on CD58 expression in their tumors revealed that patients with CD58 expression tumors had a poorer survival compared to patients with low or non-expressing tumors (median survival 64 vs 43 weeks, 2yr survival 3% vs 25%, p=0.0192, logrank). In summary, CD58 expression identifies a highly proliferative subpopulation of GSCs, improves upon established markers of these cells, and is an independent predictor of survival based on expression in patient tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2011-3313