Characterization of Patient‐Derived Glioblastoma Stem Cells for Marker Expression, Motility, and Invasiveness

Abstract

No effective treatment exists for Glioblastoma Multiforme (GBM), the most common and deadly form of brain cancer. Many studies have increasingly pointed towards Glioblastoma Stem Cells (GSCs) as the main cause of GBM malignancy, but mechanisms controlling their invasiveness are not fully understood. Our lab has shown that the L1 Cell Adhesion Molecule (L1CAM) increases GBM cell motility and proliferation through autocrine/paracrine stimulation in vitro. Our objective was to determine whether or not L1CAM plays a similar role in GSCs derived from patient surgical specimens. Our recent focus has been on characterizing our GSC lines for L1CAM expression and on developing bright fluorescently labeled GSCs for in vivo invasion experiments. Characterization of GSC lines for two accepted GSC “markers” has revealed that GSC lines isolated by our adherent culture method resulted in cells almost exclusively positive for Sox‐2 and nestin by flow cytometry. When placed in “differentiation media” containing 10% FBS, one GSC line retained stable expression of GSC markers, while another showed total loss of Sox‐2, but kept nestin. Analysis via western blot showed several GSC cell lines expressed L1CAM as well as the ADAM10 protease that cleaves L1 from the cell surface to stimulate nearby cells. GSCs infected with the K72 lentiviral vector encoding Green Fluorescent Protein (GFP) resulted in bright green fluorescent labeling. Injection of labeled GSCs was done into chick embryo brains at E5 either by themselves or mixed with other cell lines labeled with mCherry. Tumors were allowed to form for 10 days, brains were dissected and fixed at E15, and 350 um vibratome sections were mounted on slides for analysis by confocal microscopy. Analyzed were tumor formation, extent of cell invasion, and deposition of “trails” of L1CAM that might guide GSCs to be more invasive. The GSC line that exhibited stable Sox‐2 expression was highly invasive into brain, kept its stem cell morphology, and tended to form a medulla in mixed tumors with other GBM cell lines. The GSC line that lost Sox‐2 expression was also highly invasive, but many cells exhibited long processes like axons, and cells tended to form the cortex of mixed tumors with other GBM cell lines. We conclude that our GSC lines show fundamentally different invasive behavior and propensities to remain as GSCs. Furthermore, the chick embryo brain tumor model appears to allow a range of individual cell phenotypes to develop from GSC and GBM cell lines that presumably reflect their origins and, thus, it is a good model system to characterize phenotypes of patient‐derived GSC lines.Support or Funding InformationWe thank the UD Summer Scholars Program, the UD Undergraduate Research Program, Dept. of Biological Sciences Undergraduate Research Fund, and graduate student Reetika Dutt.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.