C1 treatment inhibits GSC proliferation <i>in vitro</i> and <i>in vivo</i>.

<p>A, Graph of neurosphere forming assay indicating the relative neurosphere numbers of C1-treated patient-derived GBM samples (GBM146, GBM157, and GBM206) and normal neural progenitors (16wf). B, CD133(+) and (−) cells, separated from GBM157-derived sphere cultures, were treated with 1 μM C1 or DMSO (control) under the identical serum-free conditions for 48 hours. The effect on CD133(+) cells was assessed by the neurosphere number per well, and the effect on CD133(−) cells was assessed by the % change of the total cell number in comparison to the control sample. C, Schematic showing organotypic slice cultures explanted GBM tissues and treated with C1 or DMSO (control) for 16 hours and evaluated with H&E, Ki67, and Nestin staining. D, Immunohistochemistry of C1- or DMSO-treated GBM slice cultures with anti-Ki-67 monoclonal antibody (Original magnification, ×200). E, Graph indicating the numbers of neurospheres (left) or total cells (right) in serum-free medium derived from C1- or DMSO-treated slice cultures for 16 hours. F, Schematic drawing of the effect of C1 treatment for the mouse intracranial GBM models derived from GSCs. Cells from GBM157 spheres were injected intracranially into immunocompromised mice (C1 mice: n = 4, control mice: n = 12). At day 7 post transplantation, C1 was injected intratumorally at quantities of 2.5 pmol (n = 3), 25 pmol (n = 4), or 250 pmol (n = 5). G, Representative images for immunohistochemistry with Ki-67 staining of GBM slice cultures treated with 25 pmol C1 or DMSO at day 10 of treatment. Ki-67 positive cells in each group were analyzed automated digital image analysis (Original magnification, ×200). H, Representative images for immunohistochemistry with human-specific Nestin antibody using GBM157-derived mouse intracranial tumors treated with varying doses of C1 or DMSO intratumoral injection (bar: 1 mm). I, Graph indicates tumor sizes in each group as determined by Nestin staining intensities analyzed using automated digital image analysis.