Abstract 3403: The neural stem cell marker GLAST, is involved in proliferation and invasion by glutamate release
Authors: Serena Pellegatta, Federica Pisati, Gabriele Cantini, Véronique Frattini, Pietro Luigi Poliani, Gaetano Finocchiaro
Journal: Cancer Research (2011)

Abstract The identification of antigens preferentially expressed in glioblastoma (GBM) and involved in its malignant phenotype is critical for for developing therapeutic strategies. We observed by DNA microarray analysis and confirmed by western blot and immunocytochemistry that the neural stem cells marker GLAST (Glutamate Aspartate Transporter), highly expressed in cells of the radial glia, is also expressed in the plasma membrane of murine and human GBM stem-like cells (GSC). GLAST is a membrane protein expressed by astrocytes with a relevant role in glutamate uptake. When we evaluated the uptake in different GSC lines we found that their glutamate uptake is up to 100 fold lower than in normal astrocytes. GSC are able to release glutamate in culture medium just as hypoxic astrocytes, showing that in pathological conditions such as ischemia, transporters can reverse uptake and release glutamate. We have used GLAST as a marker to isolate a GSC subpopulation by immunomagnetic sorting and test the capacity of GLAST-enriched cells for tumor formation in mice. Using the murine glioma model GL261, GLAST+ cells injected intracranially were significantly more tumorigenic than GLAST- or unsorted cells (p=0.00057 and p=0.00028, respectively). We confirmed these observations in nude mice using GSC from one human recurrent GBM. Histological analysis provided evidence of the invasive nature of GLAST+ GSC asin the contralateral hemisphere many tumor cellsexpress GLAST and the migration marker Double-Cortin. To evaluate the functional role of GLAST in GSC we decided to target GLAST expression using lentiviral particles expressing GLAST shRNA. GLAST inhibition impacts on cell proliferation and especially on Matrigel invasion in vitro and interferes with tumor progression, improving survival in injected nude mice (p = 0.0034 shGLAST vs Scrambled). As a consequence of GLAST interference we also found in vitro a significant reduction of glutamate release in silenced cells compared to control cells(non specific shRNA). We investigated the expression of GLAST in 60 human primary GBM and 15 low grade gliomas (LGG) by immunohistochemistry. We found marked expression of GLAST in the large majority of GBM, in contrast LGG were negative or showed nuclear expression of GLAST. When we correlated GLAST expression to overall survival of 51 GBM patients we found that high percentage of GLAST pos cells and moderate or strong reactivity correlate with decreased overall survival (p=0.02). Our data suggest that GLAST is expressed in GBM and may be involved in proliferation and invasion. In addition GLAST could be investigated as clinical marker in association with prognosis of GBM patients and for development of novel therapeutic strategies. 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 3403. doi:10.1158/1538-7445.AM2011-3403