Notch pathway blockade inhibits glioblastoma growth by depleting CD133 positive stem-like cancer cells

5001 CD133-positive cancer stem cells (CSCs) in glioblastoma (GBM) were recently shown to be resistant to commonly used chemo- and radiation-therapies ( Nature, 444:756-60, 2006; Mol Cancer, 5:67, 2006 ). Finding reagents that can target CSCs may therefore improve the treatment of GBM patients. It has been shown that the Notch signaling pathway regulates normal stem cells in the central nervous system, and that GBM neurosphere lines show high Notch activity ( Cancer Research, 64:7011-21, 2004; Cancer Cell, 9:391-403, 2006 ). However, it is unknown if Notch pathway blockade could target CSCs in GBM. We have demonstrated recently that Notch pathway inhibition depletes CSCs and blocks engraftment in medulloblastomas ( Cancer Research, 66:7445-52, 2006 ), a common malignant brain tumor in children. Recent studies have shown that GBM propagated as neurospheres more accurately replicate the infiltrating growth patterns seen in primary tumors when injected into nude mice, and contain stem-like cancer cells. In the present study, we used GBM neurosphere lines to examine the effects of Notch pathway inhibition on CSCs. We found that Notch pathway blockade by 2 µM levels of the Gamma-Secretase Inhibitor GSI-18 reduced expression of CD133 by more than 90% and inhibited tumor growth in 3 independent lines by 20 to 70% in vitro . In contrast, overexpression of an active form of Notch2 (NICD2) increased both pathway activity and tumor growth approximately 2-fold in vitro . When equal numbers of viable GSI-18 or vehicle (DMSO) treated GBM neurosphere cells were injected subcutaneously into nude mice, DMSO treated cells always formed tumors whereas GSI-18 treated cells did not. Most importantly, in vivo delivery of GSI-18 by polymer based beads two weeks after orthotopic tumor engraftment also effectively blocked the tumor growth intracranially and significantly prolonged survival. We believe that this compound is acting primarily via Notch blockade, as mice bearing orthotopic xenografts stably transfected with GSI-insensitive NICD2 formed larger tumors and had uniformly short survival when treated with GSI-18. These results suggest that depleting CSCs by Notch pathway blockade inhibits tumor growth and propagation both in vitro and in vivo . Finally, reduced proliferation was seen in GSI-18 treated GBM stem-like cells, as evidenced by decreased Ki-67 expression observed selectively in the Nestin+ population. Increased expression of cleaved caspase-3 was also seen in GBM neurospheres following Notch blockade. Reduced expression of phosphorelated Akt and STAT3 in these GBM neurosphere cells after GSI-18 application suggests that Notch pathway inhibition depletes CSCs through Akt/STAT3 mediated apoptosis. In summary, we demonstrate that Notch pathway blockade depletes stem-like cells in GBMs, suggesting that Gamma-secretase inhibitors can be used as chemotherapeutic reagents to target CSCs in malignant brain tumors.