Article Figures & Data
Figures
- FIGURE 1.
The tissue environment of cell implantation dictates the expression of CD133(+) brain tumor–derived stem cells. Cultured DAOY monolayers were trypsinized and 106 cells injected either subcutaneously or intracerebrally into nude mice. Tumors were left to develop as described in Materials and Methods. A. Histopathologic analysis of paraffin sections (3 μm) of intracerebrally implanted human DAOY medulloblastoma cells was done with hematoxylin-phloxin-saffron staining and shows (1) malignant proliferation composed of a majority of small round cells (arrow) found in normal mouse brain (*; magnification, ×100). At high magnification (×400), hematoxylin-phloxin-saffron staining shows (2) that tumor is highly cellular with numerous mitoses, and presents an aggressive pattern (3) with limited area of necrosis (magnification, ×200). Some tumoral cells (4) had a membranous expression of CD133 (arrows). B. Immunoblotting was used to assess CD133 protein expression in lysates (20 μg proteins) generated from subcutaneous and intracerebral U87 glioblastoma-implanted or DAOY medulloblastoma-implanted cells as described in Materials and Methods. C. Lysates (5 μg protein) from in vitro cultured DAOY cell monolayers (DAOY), contralateral healthy brain (Ctrl), and from intracerebrally implanted DAOY cells (Tumor) were assessed for CD133 expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an internal loading control. D. Relative gene expression levels of CD133 (CD133), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were assessed by quantitative PCR using total RNA extracted from contralateral healthy brain (white columns) or from intracerebrally implanted DAOY tumors (black columns).
- FIGURE 2.
Increased CD133, MDR-1, MT1-MMP, and MMP-9 expression characterize the invasive phenotype of neurosphere-like DAOY cells. A. Representative phase contrast pictures of DAOY monolayers and neurosphere-like cells were taken after 48 h of culture. B. Total RNA was extracted from DAOY monolayers and neurosphere-like cells as described in Materials and Methods. Semiquantitative RT-PCR was done and cDNA amplicons for MMP-9, HuR, CD133, MDR-1, MT1-MMP, and glyceraldehyde-3-phosphate dehydrogenase were revealed by electrophoresis using agarose gels as described in Materials and Methods. C. Conditioned medium from DAOY monolayers and neurosphere-like cells were assessed for their MMP-9 and MMP-2 secretion and activation levels as described in Materials and Methods. D. DAOY monolayers and neurosphere-like cells were trypsinized and seeded (5 × 104 cells) on gelatin-coated filters in modified Boyden chambers. Migration was allowed to proceed for 16 h at 37°C. Cells on filters were then fixed and stained; one out of five representative stainings for each condition. Columns, mean of a representative experiment in which five random fields per filter were counted for each condition; bars, SD.
- FIGURE 3.
MT1-MMP and MMP-9 control the neurosphere-like formation process in DAOY cells and contribute to their invasive phenotype. A. Gene silencing using siRNA was used to down-regulate the expression of MT1-MMP and MMP-9 in DAOY cell monolayers (top) or DAOY neurospheres (bottom) as described in Materials and Methods. Conditioned medium from each respective condition was harvested 48 h posttransfection and the extent of either MT1-MMP or MMP-9 silencing assessed using gelatin zymography. B. MT1-MMP immunoblotting was done with cell lysates isolated from A. Autoradiograms are shown at 30-s vs. 2-min expositions in order to appreciate MT1-MMP down-regulation (30 s exposition), and the appearance of the 43 to 45 kDa inactive immunoreactive MT1-MMP that is generated during proMMP-2 activation (2 min exposition). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. C. Phase contrast (magnification, ×10 and ×4) microscopy enabled the visualization of the decrease in neurosphere-like formation in cells from which MT1-MMP or MMP-9 had been silenced. D. Cell migration was also assessed 48 h posttransfection in modified Boyden chambers as described in Materials and Methods for control (white columns) monolayers and neurosphere-like DAOY cells, and cells in which MMP-9 (black columns) and MT1-MMP (gray columns) gene expression was silenced. Columns, mean of a representative experiment in which five random fields per filter were counted for each condition; bars, SD.
- FIGURE 4.
Overexpression of MT1-MMP triggers neurosphere-like DAOY differentiation. A. DAOY cell monolayers were transiently transfected with a plasmid encoding GFP-MT1-MMP as described in Materials and Methods. Representative pictures were taken in phase contrast (top) and fluorescence (bottom) in order to show transfection efficacy. B. Conditioned medium from mock and GFP-MT1-MMP–transfected cells were assessed for proMMP-2 activation by the expression of the recombinant MT1-MMP protein using gelatin zymography as described in Materials and Methods. C. Mock or GFP-MT1-MMP–transfected cells were trypsinized and then seeded either in monolayer medium or neurosphere medium. Representative pictures were then taken in phase contrast and fluorescence in order to monitor the extent of MT1-MMP–mediated neurosphere-like formation.
Volume 6, Issue 6
