Skip to main content
. Author manuscript; available in PMC: 2015 Apr 24.
Published in final edited form as: Cell. 2014 Apr 10;157(3):580–594. doi: 10.1016/j.cell.2014.02.030

Figure 3. A core TF network for tumor-propagating GBM cells.

Figure 3

(A) Data points indicate percentage of single-cell DGCs capable of forming spheres in serum-free conditions. Each of the 19 TFs in Figure 2A was tested alone (first column, ‘single TF’), in combination with POU3F2 (second column) or in combination with POU3F2 and SOX2 (third column). HLH family TFs were also tested in combination with POU3F2, SOX2 and SALL2 (fourth column), based on an enrichment of HLH motifs in regulatory elements that failed to activate in 3TF-induced DGCs. TF combinations that enhanced in vitro spherogenicity (blue) were selected for in vivo testing. (B) Flow cytometry profiles show expression of the stem cell marker CD133 for DGCs induced by the single, double, triple and quadruple TF combinations with the highest in vitro sphere-forming potential. (C) For TF combinations with in vitro spherogenic potential (blue in panel 3A), 100,000 cells were injected in the brain parenchyma (n=4 mice per TF combination). Survival curve is shown for this in vivo tumor-propagation assay. Only the quadruple TF combination POU3F2+SOX2+SALL2+OLIG2 initiated tumors in mice. (D) Tumor histopathology shows characteristic features of glioblastoma, including necrotic areas (*) and crossing of corpus callosum (boxed area). At high magnification cells show atypical features and mitotic figures are evident (arrows). LV: lateral ventricle. (E) Secondary TPC sphere cultures (“iTPC”) derived from xenotransplant tumors express the stem-cell marker CD133. (F) Contrast field image of iTPC spheres. (G) Left: bar graph shows iTPC and TPC proliferation rates measure by BrdU incorporation. Right: data points indicate percentage of single cells capable of serial sphere formation in three consecutive passages in serum-free conditions. Self-renewal properties and proliferation of iTPCs are comparable to corresponding TPCs. (H) Orthotopic serial xenotransplantation in limiting dilution shows that as few as 50 MGG8 iTPC are sufficient to initiate tumors. (I) Data points indicate in vitro sphere formation of MGG4 TPCs infected with lentivirus containing shRNA for POU3F2, OLIG2 or SALL2, compared to control (two hairpins per TF). (J) Survival curve depicts in vivo tumor propagating potential of MGG4 TPCs infected with POU3F2 shRNA, SALL2 shRNA, OLIG2 shRNA or control shRNA. See also Supplemental FiguresS2–S4.