Characterization of sphere-propagating cells with stem-like properties from DU145 prostate cancer cells.

While accumulating evidence demonstrates the existence of prostate cancer stem cells (PCSCs), PCSCs have not been isolated and thoroughly characterized. We report here the enrichment and characterization of sphere-propagating cells with stem-like properties from DU145 PC cells in a defined serum-free medium (SFM). Approximately 1.25% of monolayer DU145 cells formed spheres in SFM and 26% of sphere cells formed secondary spheres. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins (34βE12 and CK18) and for cancer stem cell markers, including CD44, CD24, and integrin α2β1. Upon culturing spheres under differentiating media conditions in the presence of 10% serum, cells positive for CD44 and CD24 were substantially reduced. Furthermore, spheres could be generated from the sphere-derived adherent cell cultures and xenograft tumors, demonstrating the stemness of DU145 spheres. We have maintained spheres for more than 30 passages within 1.5years without noticeable loss of their "stemness". Sphere cells possess self-renewal capacity, display significant increases in proliferation potential, and initiate xenograft tumors with enhanced capacity compared to monolayer DU145 cells. While EGF promoted the generation and maintenance of these stem-like cells, bFGF inhibited these events. Sphere cells proliferate slowly with a significant reduction in the activation of the PI3K-AKT pathway compared to monolayer DU145 cells. While knockdown of PTEN enhanced AKT activation, this did not affect the generation of primary spheres and the propagation of secondary spheres. Consistent with this observation, we were able to demonstrate the generation and propagation of spheres without the addition of external growth factors. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. 2011 Elsevier B.V. All rights reserved.