Abstract 3398: Neurosphere formation, multilineage differentiation and DNA damage repair potential in glioblastoma: Exploring the role of CD133
Authors: Joseph Justin V. , Balasubramaniyan V , Yuvaraj Saravanan , Kruyt Frank A.E. 
Published: June 2012

Abstract
Glioblastoma (GBM) is one of the most lethal and most aggressive forms of primary brain tumour. Despite the advances made in biomedical research the median survival rate in GBM which is 12-15 months hasn't improved significantly over the past three decades. There has been increasing amount of evidence indicating the presence of a rare population of cancer stem cells in GBM, called the glioblastoma stem cells (GBSCs). It is hypothesized that GBSCs are important in maintaining GBM and rendering them the property of drug/radiation resistance. CD133 (Prominin 1) is one of the most commonly used markers for isolating the stem cell population from GBM although debate is ongoing on the stem cell selectivity of the marker. Apart from this the physiological function of CD133 remains unknown.
The objective of this study is to address two key questions associated with CD133 in GBM: first, how effective is CD133 as a cancer stem cell marker in GBM? And second, what is the function of CD133? To examine these questions three glioblastoma cell lines, GSC-23 (provided by H. Colman, Dept. of Neurosurgery and Huntsman Cancer Institute,
University of Utah), U87 and A172 were used of which A172 cells failed to grow as neurospheres and lacked CD133 expression. Neurospheres were generated from U87 cells, despite that CD133 expression was not detectable in these cells. The CD133+ and CD133- fraction were FACS sorted from GSC-23 cells and their ability to form neurospheres and to demonstrate multilineage differentiation potentials were tested in the + and – fraction. Neurosphere formation was tested by performing limiting dilution assays in serum free medium containing bFGF and EGF. Multilineage differentiation potential was determined by culturing in 10% FCS using neuronal and glial markers such as MAP2, β3 tubulin, GFAP and O4. Both + and – fractions demonstrated neurosphere formation and differentiation potential. In addition, the capacity to repair DNA damage induced via irradiation was quantified using γH2AX staining, showing no differences in this capacity. Next, we generated retroviral vectors containing a CD133 short-hairpin (sh)RNA or the CD133 coding sequence in order to study the effect of CD133 knock-down and overexpression in glioblastoma cells derived from both established cell lines and primary material. These experiments are currently in progress. The reproducibility of the in vitro results will be evaluated further in mice models
In conclusion, our preliminary results indicate that neurosphere formation, multilineage differentiation and DNA damage repair are independent of the CD133 status in GBM cells. The possible physiological role of CD133 is presently under further investigation. This study will increase our understanding on the credibility of CD133 as a CSC marker in GBM and could also throw light on the possible functional attributes of CD133.
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 3398. doi:10.1158/1538-7445.AM2011-3398