The establishment of U87 glioma sphere cell invasion model in zebrafish embryos.

A. Dual color confocal image shows that U87 sphere cells (RFP labeled, red) were microinjected into the middle of yolk sac within Tg (fli1:EGFP)^y1 transgenic zebrafish embryos (EGFP labeled, green). B. Different numbers of U87-RFP glioma sphere cells were microinjected into Tg (fli1:EGFP)^y1 embryos (n = 300 in each group), and the percentage of embryos with invasive tumor cells was quantitated. C. The survival rate of Tg (fli1:EGFP)^y1 zebrafish embryos microinjected with different numbers of U87-RFP glioma sphere cells (n = 300 in each group). D. Representative dual color confocal images of RFP-labeled U87 sphere cells within Tg (fli1:EGFP)^y1 zebrafish embryos at the different invasive stages. Red: RFP-labeled U87 sphere cells; Green: Tg (fli1:EGFP)^y1 microvessels.

Invasive U87 sphere cells express CD133.

A. U87 sphere cells with various invasion capability within zebrafish embryos. The extent of invasion was classified in three degrees: Low: less than 5 migrated cells; Medium: 5–20 migrated cells; High: more than 20 migrated cells. Representative images at higher magnification show the invasive RFP-labeled U87 sphere cell masses (red) in the tail region of the embryos via EGFP-labeled host vessels (green). B. Detection of CD133 expression on non-invasive and invasive U87 sphere cells at 2 dpi by immunofluorecent staining. All of U87 sphere cells within injected embryos were stained with monoclonal anti-CD133 antibody (1:300) and examined by confocal microscopy. Green: Tg (fli1:EGFP)^y1 microvessels; red: RFP-labeled U87 sphere cells; blue: CD133 positive U87 cells. C. Quantitative analysis of CD133-expressing cells in non-invasive cell group (n = 713) and high-invasive cell group (n = 175) at 2 dpi. (p<0.001).

CD133+ U87 GSCs are highly invasive within zebrafish embryos.

A. Representative images of the invasion of differentiated U87 cells, U87 sphere cells, and CD133+ U87 GSCs within the injected embryos at 2 dpi. The images at higher magnification show the invasive RFP-labeled cell masses at tail region of embryos via host vessels. B. The percentage of the embryos with invasive cells injected with RFP-labeled differentiated U87 cells, U87 sphere cells, and CD133⁺ U87 GSCs. The data were obtained from three replicate experiments of 50 injected embryos in each experiment: n = 124 for live embryos injected with differentiated U87 cells, n = 121 for embryos injected with U87 sphere cells, and n = 120 for embryos injected with CD133⁺ cells C. The percentage of invasive cells within total injected cells (Invasion Index) in the embryos. All injected cells including invasive or non-invasive cells within zebrafish embryos were evaluated by ImageJ software through fluorescence intensity. n = 37200 (300 injected cells per embryo among 124 live embryos) for differentiated U87 cell group, n = 36300 (300 injected cells per embryo among 121 live embryos) for U87 sphere cell group, and n = 36000 (300 cells per embryo among 120 live embryos) for CD133⁺ U87 GSCs group (p<0.001).

CD133 expression on GSCs is reduced after spread in zebrafish embryos.

A. CD133 expression examined on non-invasive or invasive cells in the embryos injected with CD133⁺ U87 GSCs. All CD133⁺ U87 GSCs within injected embryos were stained with a monoclonal anti-CD133 antibody (1:300) and examined by confocal microscopy. Green: EGFP-labeled endothelial cells; red: RFP-labeled U87 sphere cells; blue: CD133⁺ U87 GSCs. B. The percentage of CD133⁺ cells in non-invasive cells or invasive cells at 2 dpi (n = 300 each group). C. Detection of CD133⁺ cell percentage in sorted CD133⁺ U87 cells in preparation for microinjection by flow cytometry (p<0.001).