br Experimental Procedures br Acknowledgments

Experimental Procedures
Acknowledgments This work was supported by BONFOR/Gerok Scholarships O-137.0015 (to B.V.S.), Ruediger Foundation grants (to B.V.S.), State Scholarship Fund/Chinese Scholarship Council 2008627116 (to Z.L.), NIH – National Eye Institute 5R01EY022079-02 (to S.T.), and NYSDOH contract C028504 (to S.T. and J.H.S.) supported by the Empire State Stem Cell Fund. Opinions expressed here are solely those of the authors and do not necessarily reflect those of the National Institutes of Health, Empire State Stem Cell Board, the New York State Department of Health, or the state of New York. A European patent application on the shooter instrument was submitted by B.V.S., Z.L., R.B., N.E. and F.G.H. (PCT/EP2012/058083). S.T. and J.H.S. have a granted US patent on aRPESC (application number 12/428,456). This paper was presented in part at the 20th ISER meeting in July 2012 and the 3rd TERMIS World congress in September 2012. C. Strack assisted with histologic processing, and J. Bedorf at the Institute of Pathology, University of Bonn was very helpful in obtaining transmission electron micrographs. Dr. Andrea Lohmer of HET facility, University Clinics Bonn, Bonn, Germany is gratefully acknowledged for her veterinarian services. N. Braun of Geuder AG Heidelberg, Germany provided support for instrumentation used in rabbits. We thank Carol Charniga for invaluable help with RPE dissections and culture maintenance.
Introduction Recent studies have provided strong support for the cancer stem cell (CSC) hypothesis, which suggests that many cancers, including breast cancer, are driven by a subpopulation of DZNep that display stem cell properties. These cells may mediate metastasis and, by virtue of their relative resistance to chemotherapy and radiation, contribute to treatment relapse. Although some studies have indicated a close association between CSCs and the acquisition of an epithelial-mesenchymal transition (EMT) state (Mani et al., 2008), other studies have suggested that EMT and CSC states are mutually exclusive (Tsuji et al., 2008). The process of EMT plays an important role in embryogenesis as well as in a number of biological processes associated with cancer progression (Thiery et al., 2009). During EMT, epithelial cells lose cell-cell contacts, undergo cytoskeletal remodeling resulting in loss of polarity, and acquire a mesenchymal morphology (Moreno-Bueno et al., 2008). Importantly, EMT is reversible, and the epithelial phenotype generated through mesenchymal-epithelial transition (MET) is characterized by expression of E-cadherin and establishment of cell polarity. Interestingly, a number of pathways that are known to regulate CSCs, including Notch, hedgehog, Wingless (Wnt), transforming growth factor-β (TGFβ), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), also are capable of inducing EMT (Shin et al., 2010; Takebe et al., 2011; Yoo et al., 2011). However, other pathways that regulate CSCs, including those involving bone morphogenetic proteins (BMPs) and human epidermal growth factor receptor (HER) signaling, promote MET (Korkaya et al., 2012; Samavarchi-Tehrani et al., 2010). Further studies are needed to more fully define the relationship among EMT, MET, and CSCs. The development of biomarkers to identify BCSCs by our group and others, as well as validation of in vitro and mouse models, has facilitated the isolation and characterization of BCSC from both murine and human tumors (Al-Hajj et al., 2003; Dontu et al., 2003; Ginestier et al., 2007). In human breast cancer, tumor-initiating cells were first identified by virtue of their expression of the cell surface marker profile CD24−CD44+. In primary breast xenografts, cells expressing these markers were enriched for their ability to initiate tumors in immunodeficient nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (Al-Hajj et al., 2003). More recently, we have shown that both normal and malignant breast stem cells that express the enzyme aldehyde dehydrogenase (ALDH), as assessed by the ALDEFLUOR assay, are also enriched for tumor-initiating characteristics (Ginestier et al., 2007). Furthermore, in primary breast xenografts, CD24−CD44+ and ALDH identified overlapping, but nonidentical cell populations, each capable of initiating tumors in NOD/SCID mice (Ginestier et al., 2007). Tumor cells that simultaneously expressed both CSC markers (i.e., CD24−CD44+ and ALDH+) displayed the greatest tumor-initiating capacity, generating tumors in NOD/SCID mice from as few as 20 cells (Ginestier et al., 2007). Subsequently, CD44, CD24, and ALDH were reported to be expressed in CSCs from a wide variety of carcinomas, including those of the pancreas, colon, lung, ovary, and prostate gland (Eramo et al., 2008; Huang et al., 2009; Kryczek et al., 2012; Li et al., 2007; Prince et al., 2007). In addition to carcinomas, these markers have also proven useful for isolating CSCs from hematologic malignancies (Storms et al., 1999) and sarcomas. This suggests that CSCs across a wide variety of malignancies may share marker expression as well as biological characteristics. However, it remains unclear whether tumors contain multiple types of CSCs and whether CSC markers identify distinct CSC populations.