ORIGINAL ARTICLE HighlypurifiedCD44 þ prostatecancercellsfromxenografthumantumors are enriched in tumorigenic and metastatic progenitor cells L Patrawala 1 , T Calhoun 1 , R Schneider-Broussard 1 , H Li 1 , B Bhatia 1 , S Tang 1 , JG Reilly 1 , D Chandra 1 , J Zhou 1,3 , K Claypool 1 , L Coghlan 1 and DG Tang 1,2 1 Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, TX, USA and 2 Program in Environmental & Molecular Carcinogenesis, Graduate School of Biomedical Sciences (GSBS), Houston, TX, USA CD44isamultifunctionalproteininvolvedincelladhesion andsignaling.TheroleofCD44inprostatecancer(PCa) development and progression is controversial with studies showing both tumor-promoting and tumor-inhibiting effects. Most of these studies have used bulk-cultured PCa cells or PCa tissues to carry out correlative or overexpression experiments. The key experiment using prospectively purified cells has not been carried out. Here weuseFACStoobtainhomogeneousCD44 þ andCD44  tumor cell populations from multiple PCa cell cultures as well as four xenograft tumors to compare their in vitro and in vivo tumor-associatedproperties.Ourresultsreveal that the CD44 þ PCa cells are more proliferative, clonogenic, tumorigenic, and metastatic than the isogenic CD44  PCa cells. Subsequent molecular studies demon- strate that the CD44 þ PCa cells possess certain intrinsic properties of progenitor cells. First, BrdU pulse-chase experiments reveal that CD44 þ cells colocalize with a population of intermediate label-retaining cells. Second, CD44 þ PCa cells express higher mRNA levels of several ‘stemness’ genes including Oct-3/4, Bmi, b-catenin, and SMO.Third,CD44 þ PCacellscangenerateCD44  cells in vitro and in vivo. Fourth, CD44 þ PCa cells, which are AR  , can differentiate into AR þ tumor cells. Finally, a very small percentage of CD44 þ PCa cells appear to undergo asymmetric cell division in clonal analyses. Altogether, our results suggest that the CD44 þ PCa cell population is enriched in tumorigenic and metastatic progenitor cells. Oncogene (2006) 25, 1696–1708. doi:10.1038/sj.onc.1209327; published online 30 January 2006 Keywords: CD44; prostate cancer; progenitor cells; cancer stem cells; tumor progenitor cells; stemness genes Introduction Human prostatic glands consist of two major epithelial cell types: basal and secretory (luminal). Basal cells express cytokeratin 5 (CK5), CK14, Bcl-2 (McDonnell et al., 1992), Her-2/neu (Reiter and Sawyers, 2001), p63 (Signoretti et al., 2000), and CD44 (Liu et al., 1997), whereas differentiated luminal cells express CK8, CK18, AR (androgen receptor), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), CD57, and 15-lipoxygenase 2 (Tang et al., 2002; Bhatia et al., 2005). The basal cell compartment is thought to contain stem/ progenitor cells as >80% of the proliferating cells are localized in the basal layer (Bonkhoff et al., 1994). Furthermore, molecules known to play an important role in maintaining stem/progenitor cell proliferation (e.g. p63), survival (e.g. Bcl-2), and detoxification (e.g. GST-p), all localize preferentially in the basal cell compartment. That the adult human prostate contains stem cells (SCs) is also supported by the observation that a small number of cells within the prostate poss- ess tremendous proliferative capacity and can form glandular-like structures in reconstituted systems (Hud- son et al., 2000). Several populations of putative human prostate stem/progenitor cells have been reported, which include CK5 and CK18 double-positive (CK5 þ /CK18 þ ) intermediate cells (van Leenders et al., 2000; Wang et al., 2001; Tran et al., 2002; Garraway et al., 2003; Bhatia et al., 2005), the side population (SP) cells (Bhatt et al., 2003), and small populations of cells that preferentially express cell surface molecules CD44 (Liu et al., 1997), a2b1 (Collins et al., 2001), or CD133 (Richardson et al., 2004). CD44, which is spliced into many isoforms, is an adhesion molecule with multiple signaling functions (Ponta et al., 2003). In the normal prostate, CD44 is expressed in most basal cells. In prostate cancer (PCa), CD44 expression varies in different studies. One study of 109 cases reports a complete lack of membranous expression of all CD44 isoforms in 93–98% of primary PCa examined (Kallakury et al., 1996). However, another study of 74 PCa lesions reports moderate to high levels of CD44 expression in 60% of primary tumors with B14% of metastases expressing low levels of CD44 (Nagabhushan et al., 1996). Yet another study Received 9 September 2005; revised 10 November 2005; accepted 11 November 2005; published online 30 January 2006 Correspondence: Professor DG Tang, Department of Carcinogenesis, Science Park-Research Division, The University of Texas MD Anderson Cancer Center, 1808 Park Road 1C, Smithville, TX 78957, USA. E-mail: dtang@mdanderson.org 3 Current address: Dermatology Branch, National Cancer Institute, NIH, Building 10, Room 12N262, 10 Center Drive, MSC 1908, Bethesda, MD 20892-1908, USA. Oncogene (2006) 25, 1696–1708 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc