Abstract 257: In vitro tumorigenicity and stemness characterization of the U87MG glioblastoma cell line: Monolayer cultures, neurospheres, and CD133+ and CD133- sorted fractions.
Authors: Vacas-Oleas A , de la Rosa J , Garcia-Lopez R , Vera-Cano B , Gallo-Oller G , Alfaro M , Shahi M H. , Fan X , Encio I J. , Rey J A. , Castresana Javier S. 
Published: August 2014

Abstract
The cancer stem cells (CSC) hypothesis is nowadays the most widely accepted theory regarding tumor formation and self-renewal capacity. It states that one of the different tumorigenic phenotypes inside a tumor mass is capable of generating new tumors if transplanted onto a host and it is able to self-generate and regenerate the rest of the tumor cells. The necessity to find pharmacological compounds or biological agents capable of eliminating CSC makes the search for methods of recognition and isolation of these cells a matter of great urgency.
Our working hypothesis is that the selection of cancer cells with the CD133 marker and their subsequent isolation and characterization will allow us to assess the capacity of the different CD133 subpopulations as CSC. The aim of the study was to separate CD133+ and CD133- cell subpopulations from the U87MG glioblastoma cell line by magnetic activated cell sorting (MACS), and to analyze the tumorigenic and stem cell differences among four sets of cells: the cell line grown in serum (monolayer group), the neurospheres of the same cell line, and the CD133+ and CD133- sorted cell fractions from the neurospheres.
Our results showed that the neurospheres and the CD133+ cells overexpressed stem cell marker genes like SOX2, Nestin, Musashi1 and CD133, and formed more colonies in soft agar than the cell groups with less CD133 expression (monolayer and CD133- group). On the other hand the CD133- and the monolayer groups had similar expression levels of CD133, while the CD133- cells expressed higher levels of Nestin, SOX2 and Musashi1 than the monolayer cells. Besides, cell migration assessed by the scratching test showed that the CD133- and the monolayer cells migrated more than the neurospheres and the CD133+ cells. Moreover, the CD133- cells had a higher proliferation rate than the CD133+ cells and the neurospheres, being all these three groups cultured in the medium for neurospheres.
In conclusion, we might point to the presence of CSC in the CD133- group, as previously suggested by others. Then, CD133+ cells are not necessarily equivalent to CSC. Maybe CD133+ cells present a higher probability of including CSC than CD133- do. Therefore, further functional and genetic analyses must be performed to reach an optimal isolation of CSC.
Citation Format: A Vacas-Oleas, J de la Rosa, R Garcia-Lopez, B Vera-Cano, G Gallo-Oller, M Alfaro, M H. Shahi, X Fan, I J. Encio, J A. Rey, Javier S. Castresana. In vitro tumorigenicity and stemness characterization of the U87MG glioblastoma cell line: Monolayer cultures, neurospheres, and CD133+ and CD133- sorted fractions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 257. doi:10.1158/1538-7445.AM2013-257