Breast cancer is the most common malignancy in United States women, accounting for >40,000 deaths each year. These breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Using a model in which human breast cancer cells were grown in immunocompromised mice, we found that only a minority of breast cancer cells had the ability to form new tumors. We were able to distinguish the tumorigenic (tumor initiating) from the nontumorigenic cancer cells based on cell surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44+CD24−/lowLineage− in eight of nine patients. As few as 100 cells with this phenotype were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. The tumorigenic subpopulation could be serially passaged: each time cells within this population generated new tumors containing additional CD44+CD24−/lowLineage− tumorigenic cells as well as the phenotypically diverse mixed populations of nontumorigenic cells present in the initial tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the elucidation of pathways that regulate their growth and survival. Furthermore, because these cells drive tumor development, strategies designed to target this population may lead to more effective therapies

Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.

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Metabolism of oxygen, while central to life, also produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease, and aging. It has recently been shown that central nervous system stem cells 1, 2 and hematopoietic stem cells and early progenitors [3][4][5][6] contain lower levels of ROS than their more mature progeny and that these differences appear to be critical for maintaining stem cell function. We hypothesized that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature # To whom correspondence should be addressed. mfclarke@stanford.edu. * Contributed equally.Author Contributions M.D. and R.W.C. contributed equally to this work. M.D, R.W.C., N.L., T.K., M.J.D., A.K., D.Q., J.S.L., L.A., and M.W. performed the experiments. B.J., M.J.K, I.W., F.W., G.S., C.G., B.P., J.S., and S.K.L. aided in human tumor tissue acquisition. G.S. designed a pre-operative protocol allowing for tissue acquisition. M.D., R.W.C., and M.F.C. designed the experiments and wrote the manuscript. S.R.Q., J.M.B., and I.L.W. provided intellectual input and aided in experimental design.Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Given the conservation of low ROS levels in several types of normal tissue stem cells, we hypothesized that CSCs in some tumors may also contain lower concentrations of ROS than their non-tumorigenic progeny. In order to investigate ROS biology in human CSCs, we began by examining the expression of genes involved in ROS metabolism in primary human breast CSCs and NTCs. Using microarray data from human breast CSC-enriched populations and NTCs 13 and a curated list of genes involved in ROS metabolism 5 (see methods), Gene Set Enrichment Analysis (GSEA) 14 revealed that the expression of ROS genes was highly overrepresented in the CD44 + CD24 -/low Lin -breast CSC-enriched population compared to NTCs (p<0.001; Supplementary Fig. S2). The ROS genes identified as the core enriched genes by GSEA included a number of important antioxidant genes (Supplementary Table 2). Thus, gene expression profiles of human breast CSC-containing populations suggest that they contain higher levels of antioxidant defense systems than NTCs. NIH Public AccessNext, we directly assessed ROS levels in human tumor subpopulations. To do this the CD44 + CD24 -/low Lin -breast CSC-enriched population and the corresponding "Not CD44 + CD24 -/low " Lin -NTC population were purified from surgically resected breast tumors ( Supplementary Fig. S3). DCF-DA staining revealed that the CSC-enriched population in the human breast tumors we examined contained significantly lower levels of prooxidants than the NTC population. In some breast tumors, the vast majority of cells in the CSC-containing fraction displayed a low ROS phenotype compared to NTCs (Fig. 1e) while ...

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