Additional Supporting Information may be found in the online version of this article.

SC_12-0327_sm_SupplFig1.tif203KFigure S1: The quantification of endothelial cell migration and invasion in the presence of conditioned-medium of TIC-high and TIC-low fractions. Endothelial cells which invaded the bottom filter of the modified Boyden chamber assay presented in Figure 2A-B, were counted, and the number of cells per field is presented for migration (upper graph) and invasion (lower graph). (n>15 fields/group).*, 0.05>p>0.01; **, 0.01>p>0.001; ***, p<0.001.
SC_12-0327_sm_SupplFig2.tif343KFigure S2: Bevacizumab does not inhibit microvessel sprouting of murine aortic ring assay. M199 BT-203 medium known to induce microvessel sprouting of aortic ring was used in the presence or absence of 5 μg/ml bevacizumab. Images of microvessel sprouting from aortic rings were captured using light microscopy (Scale bar=50μm).
SC_12-0327_sm_SupplFig3.tif274KFigure S3: The effects of chemotherapy in combination with antiangiogenic drug on volume and microvessel density of either U-87MG or PANC1 tumors. Five million of either U-87MG or PANC1 cells were subcutaneously injected into 8-10 week old nude mice (n=3-5 mice/group) when tumors reached a size of 500mm3 treatment with paclitaxel, gemcitabine, B20 antiangiogenic drug, or the combination of paclitaxel+B20 and gemcitabine+B20 was initiated. Four days later, (A) tumors were measured for the evaluation of the percentage of changes in tumor volume (after normalization to control untreated group) for either U-87MG or PANC1 tumors. Subsequently, tumors were removed and sections were immunostained for CD31 (in red) to evaluate microvessel density (micrographs are presented in Figure 4A), and (B) quantification of the number of vessel structure per field are presented for both U-87MG and PANC1 tumors *, 0.05>p>0.01; **, 0.01>p>0.001; ***, p<0.001.
SC_12-0327_sm_SupplFig4.tif199KFigure S4: VEGF-A expression in TIC-high and TIC-low conditioned medium. The level of VEGF-A as part of the analysis of different angiogenic-related factors (Figure 5) in conditioned-medium of cells from TIC-high and TIC-low fractions of either U-87MG, HT29, A549, MCF7 or PANC1 cells was evaluated using specific human VEGF-A ELISA. **, 0.01>p>0.001; ***, p<0.001.
SC_12-0327_sm_SupplFig5.tif394KFigure S5: Generation and validation of TICs from U-373MG and A172 glioblastoma cultured cells. (A) Representative images of formed tumorspheres in suspension from a series of human glioblastoma cell lines: U-373MG and A172, captured by x200 objective-field. (B) Representative flow cytometry plots of phenotypic evaluation of TIC-low and TIC-high fractions from the glioblastoma cell lines. (C) Representative flow cytometry plots for the analysis of ALDH enzymatic activity in TIClow and TIC-high fractions in the presence of ALDH substrate (BAAA) (left and middle plots, respectively) and in the TIC-high fraction in the presence of ALDH substrate and the ALDH enzyme inhibitor diethylaminobenzaldehyde (DEAB) (right plots). The percentages shown in the polygons represent the ALDH-positive population after gating on viable cells using 7AAD staining. (D) Cells from TIC-high and TIC-low fractions of the indicated cell lines were exposed to 100nM paclitaxel (PTX), 10nM gemcitabine (GEM), and cell viability was evaluated using the AlamarBlue assay. Results were plotted as fold increase from untreated cells. The generation and validation of TICs of glioblastoma cultured cells were obtained for experiments conducted in Figure 6. *, 0.05>p>0.01; **, 0.01>p>0.001.
SC_12-0327_sm_SupplFig6.tif522KFigure S6: Endothelial cell migration in the presence of conditioned medium from glioblastoma TIC-high and TIC-low fractions. (A) The migratory effect of conditionedmedium obtained from TIC-low and TIC-high fractions of glioblastoma cells was assessed by the modified Boyden chamber assay. Images were captured per x200 objective-field. (B) Endothelial cell migration was quantified by counting the number of invading endothelial cells to the bottom filter of the Boyden chamber. The number of cells per field is presented (n>15 fields/group). The evaluation of the angiogenic properties of glioblastoma cultured cells were obtained for experiments conducted in Figure 6. **, 0.05>p>0.01; ***, p<0.001.
SC_12-0327_sm_SupplFig7.tif40KFigure S7: Inhibition of VEGF-A in TIC-high fraction of glioblastoma cells. Conditioned-medium of cells from TIC-high fractions of either U-87MG or A172 cells transfected with shRNA for VEGF-A was evaluated for VEGF-A expression by specific ELISA, for experiments performed in Figure 6C-D.
SC_12-0327_sm_SupplMaterials.pdf103KSupplementary data

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