Overexpression of CXCR4 inprostate cancer progenitors.

<p>(A) Cells grown under sphere forming conditions showed an increased expression level of CXCR4 as analyzed by RT-PCR and Western blot analysis. For sphere formation, single cells were plated at 500 cells/mL in 10-cm dishes with an ultralow attachment surface and grown in serum-free epithelial basal medium for 7 days. (B) Flow cytometry analysis showed significant enrichment of the CXCR4⁺ population within CD44⁺/CD133⁺ cells compared to the total cell population for DU145 and PC3 cells (p<0.001). The cells were triple stained and analyzed with a BD LSR II flow cytometer. (C) Representative fluorescent images of CD133 and CD44 co-immunostaining showing that CXCR4⁺ DU145 and PC3 cells have a higher proportion of CD44⁺/CD133⁺ cells compared to CXCR4⁻ cells. Cells expressing high of low levels of CXCR4 were FACS-purified, plated in 384 well black clear bottom plates at a density of 100 cells/well in serum-free epithelial basal medium. After 18 hours, the cells were fixed with 3.7<b>%</b> formaldehyde in PBS and stained with anti-CD133 and anti-CD44 antibodies. Cells in at least five randomly selected fields of view were counted for each condition. Arrows show the triple positive cells. Scale bars indicate 15 µm. *- p value<0.05. (D) Immunostaining of paraffin-embedded sections of xenograft tumors formed by FACS purified CD44⁺/CD133⁺ and CD44⁻/CD133⁻ cells showed more than 13% CXCR4⁺ cells in tumors derived from CD44⁺/CD133⁺ cells compared to 2.2% CXCR4⁺ cells in xenograft tumors derived from CD44⁻/CD133⁻ cells. A total of 10³ FACS-sorted DU145 CD133⁺/CD44⁺ or CD133⁻/CD44⁻ cells embedded in BD matrigel were injected s.c. into NOD/SCID mice. Tumors were allowed to grow for 42 days until the tumors produced by DU145 CD133⁺/CD44⁺ reached a size of 400 mm³ and the tumors produced by DU145 CD133⁻/CD44⁻ reached a size of 125 mm³. Cells in at least five randomly selected fields of view were counted for each condition. Scale bars indicate 30 µm. (E) CXCR4 immunostaining on paraffin-embedded sections of xenograft tumors made by the cells grown under sphere forming and monolayer conditions showed more than 6% CXCR4⁺ cells in sphere-derived tumors as compared to 1.4% of CXCR4⁺ cells in monolayer-derived xenograft tumors. **- p value<0.01.Scale bars indicate 30 µm.