The evolution of a normal cell into a fully transformed one requires the deregulation of multiple cellular processes (1, 2). According to classic models of carcinogenesis, these events can occur in any cell. In contrast, the cancer stem cell (CSC) hypothesis holds that the preferential targets of oncogenic transformation are tissue stem or early progenitor cells that have acquired self-renewal potential (3–6). These tumor-initiating cells or CSCs, in turn, are characterized by their ability to undergo self-renewal, a process that drives tumorigenesis and differentiation, which contributes to tumor cellular heterogeneity. Recent evidence supporting the CSC hypothesis has been generated using xenografts of primary human tumors. These studies have suggested that tumors are composed of a cellular hierarchy driven by the CSC component. In addition, recent data suggest that immortalized cell lines derived from both murine and human tissues may also contain a cellular population displaying stem cell properties. Most of these studies have been based on in vitro properties, including clonogenic potential, sphere formation, and multilineage differentiation potential (7–10). More limited studies using functional transplantation of immortalized cell lines in xenografts have also suggested the existence of such a hierarchy. These studies have generally used Hoechst dye exclusion to identify the so-called side population (SP; refs. 7, 9, 11). In addition, cell surface markers defined using primary tumor xenografts, such as CD44 and CD133, have also been used to identify similar populations in established cell lines (7, 8). However, the limitations of these techniques have precluded their application across a wide variety of cell lines representing the molecular heterogeneity of tumors such as breast cancer. In addition, a crucial question remains as to whether the stem cell components of cell lines represent a valid model for CSC biology.

To provide more definitive evidence for the existence of CSC populations within breast cancer cell lines, we have studied the expression of the stem cell marker aldehyde dehydrogenase (ALDH) in a series of 33 cell lines derived from human breast cancers and nontransformed breast cells. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and is thought to play a role in stem cell differentiation through metabolism of retinal to retinoic acid (12). ALDH activity, as assessed by the fluorescent ALDEFLUOR assay, has been successfully used to isolate CSCs in multiple myeloma and acute myeloid leukemia, as well as from brain tumors (13, 14). We recently showed that ALDH activity can be used to isolate a subpopulation of cells that display stem cell properties from normal human breast tissue and breast carcinomas (15). We now show that the majority of breast cancer cell lines contain an ALDEFLUOR-positive population with a distinct molecular profile that displays CSC properties. These studies have important implications for the interpretation of data using cell lines and suggest that these lines may be useful for elucidating CSC regulatory pathways.