Our group demonstrated a role for Hh signaling in the self-renewing cells of MM and ALL. Flow cytometric analysis of MM cell lines following treatment selleck chemical with the Smo inhibitor cyclopamine resulted in decreases in the CD138-negative cell fraction (the CSC population) as well as in side population (SP) cells. To assess the effects of Hh inhibition on self-renewal potential, an in vitro clonogenic assay was performed. In this assay, cells were treated with an anti-Shh antibody 5E1 to block ligand-dependent Hh signaling or the Smo inhibitor cyclopamine and then assayed for the ability to form tumor colonies in semi-solid media. Compared to a vehicle control, both 5E1 or cyclopamine significantly inhibited colony-formation by MM cell lines and primarily clinical samples.

20 Furthermore, this inhibitory effect was maintained during serial replating indicative of self-renewal potential. Similarly, in ALL, treatment of human ALL cell lines with the Smo inhibitors cyclopamine or IPI-926 resulted in decreased ALDH+ cells, decreased in vitro colony-formation, and decreased serial transplantation in mice, all reflective of effects on the self-renewing cell population.15 In chronic myeloid leukemia (CML) mouse models, Smo ?/? mice had longer latency to develop CML when mouse bone marrow transduced with BCR-ABL was transplanted. A decrease in the number of phenotypically primitive cells (Lin- Sca-1- c-Kit+), a surrogate for CSC, was also observed in the Smo ?/? mice as compared to Smo +/+ mice.67 Similarly, Dierks et al transduced Smo ?/? fetal liver cells with BCR-ABL and observed more rapid development of CML in transplanted mice.

Secondary recipients of Smo ?/? cells had no development of CML at 9+ months of observation. CML stem cells were isolated by flow cytometry and treated with the Smo inhibitor cyclopamine or Hh ligand blocking antibody 5E1. Decreased colony-formation in vitro was observed with both treatments, demonstrating a role for ligand-dependent Cilengitide Hh signaling in mediating self-renewal in CML. Mice transplanted with BCR-ABL infected hematopoietic stem cells were treated with cyclopamine. All untreated animals rapidly developed CML and died within 4 weeks, but cyclopamine-treated mice had a 60% survival at 7 weeks. In addition, CML from cyclopamine treated mice was unable to be serially transplanted, consistent with an observed decrease in the number of CML stem cells persisting after treatment.13 In glioblastoma multiforme, elevated Gli1 expression was seen in five of 19 primary samples and four of seven cell lines tested. Treatment with cyclopamine depleted the putative CSC population as identified by increased ALDH expression and SP.