ASMA+ myoepithelial-like cells. Indeed, some ICP cells generate only CD44+ stem/progenitor-like cells and never CD442, CD24+, CK19+, ER+, or ASMA+ cells. These findings indicate that ICP cells have limited differentiation ability, at least under in vitro conditions. The differentiation ability of ICP cells appears to be lost completely or partially, so that they do not differentiate fully while they self-renew as stem/progenitor-like cells. Fourth, SCP cell lines form the same molecular cluster with luminal A and ‘‘normal-like’’ breast tumors. This suggests that SCP cell lines are phenocopies of these relatively benign and/or anti-estrogen- responsive tumors. The poor tumorigenicity of SCP cells in nude mice correlates with better tumor-free and metastasis-free survival of patients with luminal A type tumors. In contrast, ICP cell lines cluster with luminal B and basal-like breast tumor subtypes, and they are more tumorigenic, as expected for luminal B and basal- like tumor cells. It is presently unknown whether breast tumor subtypes that cluster with SCP or ICP cell lines are also composed of either differentiating or mostly self-renewing stem/progenitor cells. Recent studies reported that breast tumors may contain only CD44+, or only CD24+ cells, as well as mixed cell populations, and that CD44+ tumor cells express many stem-cell markers [27,32]. In addition, an association between basal-like breast cancer and the presence of CD44+/CD242 cells has been established [32]. The mechanisms of the differentiation block observed in ICP cell lines are not known. One might argue that cell lines that produce only CD44+, but never differentiation marker-positive cells, cannot be defined as stem/progenitor cells. However, such cell lines are not completely inert to differentiation stimuli, and may undergo differentiation under special conditions. For example, MDA-MB-231 and MDA-MB-468 cells (identified as ICP cell lines here) can be induced to differentiate into ER+ cells by Wnt5a treatment, and MDA-MB-231 cells then become sensitive to tamoxifen [33]. Most of the findings reported here are derived from in vitro studies performed with established cancer cell lines. Presently, it is unknown to what extent these findings are also relevant for breast tumors. We provide here some promising data that supports in vivo relevance of our conclusions. First, our cluster analyses associated SCP cell types with luminal A and ‘‘normal-like’’ breast tumors, whereas ICP cell types shared similar gene expression profiles with luminal B and basal-like breast tumors. Second, ICP- type MDA-MB-231 cells were more tumorigenic than SCP-type T47D cells. T47D cells formed smaller tumors in some animals, and all tumors displayed regression between 10 and 15 days post- injection. Accordingly, we observed positive SABG staining in two out of four T47D tumors, but not in MDA-MB-231 tumors. Obviously, ICP-like and SCP-like tumors in affected women may or may not display similar tumorigenic potentials, depending on the women’s hormonal status and treatment conditions. However, as most SCP-like luminal A or ER+ tumors are successfully treated with tamoxifen [34], their less aggressive behavior could be related to their highly effective senescence response to tamoxifen treatment, as shown here with T47D cells under in vitro conditions (Fig. 3). It will be interesting to examine whether the success of anti-estrogenic treatments is indeed associated with senescence induction in breast tumors. If this is the case, senescence-inducing treatments could be considered for breast cancer. In conclusion, our analyses reveal that the in vitro ability to generate senescent progeny permits discrimination between cells that share molecular and tumorigenic similarities with luminal A subtype breast tumors from cells related to basal/luminal B subtype tumors. We also provide in vitro evidence for classifying Senescence in Breast Cancer breast cancers into two major groups based on the ability to generate differentiated progeny. Less-tumorigenic SCP cell lines generate both luminal- and myoepithelial-like cells. In contrast, more-tumorigenic ICP cell lines are defective in their ability to generate differentiated progeny. Our findings may have prognostic relevance and serve as a basis for therapeutically inducing differentiation and senescence in breast cancer. Supporting Information Dataset S1 STR analysis data that shows the authenticity of breast cancer cell lines used in this study. Found at: doi:10.1371/journal.pone.0011288.s001 (1.50 MB XLS) Table S1 Gene clusters, genetic mutations and epigenetic changes of breast cancer cell lines used in this study. Found at: doi:10.1371/journal.pone.0011288.s002 (0.05 MB DOC) Table S2 Estrogen receptor (ER) status, main pathological features of senescence staining (SABG) of breast tumors used in this study. Found at: doi:10.1371/journal.pone.0011288.s003 (0.05 MB DOC) Figure S1 p16Ink4a expression in colonies obtained from breast cancer cell lines. There was no correlation between p16Ink4a expression and progenitor subtype. Found at: doi:10.1371/journal.pone.0011288.s004 (3.39 MB TIF) Figure S2 Co-staining experiments indicate that SABG staining is associated with p21Cip1, but with p16Ink4a expression in SCP cells. Colonies were generated from T47D and MB-MDA-231 cells and subjected to SABG staining, followed by p21Cip1 or p16Ink4a immunoperoxidase (brown) staining. MDA-MB-231 cells were used as negative control. Found at: doi:10.1371/journal.pone.0011288.s005 (2.74 MB TIF) Figure S3 Effect of estrogen receptor-overexpression on the production of BrdU-negative terminally arrested cell progeny. ER- overexpressing (ER-5, ER-7, ER-26) and control (C-8, C-10, C- 11) stable clones were established from T47D cells. Following transfection with ER expression and control vectors, colonies were generated from respective cell lines, labeled with BrdU for 24 h, immunostained for BrdU (brown), and slightly counterstained with hematoxylin to visualize BrdU+ and BrdU- cells. Found at: doi:10.1371/journal.pone.0011288.s006 (6.57 MB TIF) Figure S4 Detection of SABG+ senescent cells in estrogen receptor-positive breast tumors. Snap-frozen tumors were used to obtain 6 m thick sections and used directly to detect SABG+ cells. H&E: hematoxylin-eosin staining. Found at: doi:10.1371/journal.pone.0011288.s007 (9.45 MB TIF) Figure S5 ASMA+ myoepithelial-like cells are produced fre- quently in senescent cell progenitor T47D and MCF-7 cell lines under confluent conditions. ASMA was tested by immunoperox- idase. Found at: doi:10.1371/journal.pone.0011288.s008 (4.08 MB TIF) Figure S6 Unsupervised hierarchical clustering of breast tumor and cell line gene expression data that is described in Fig. 7A. Dendrogram shown here includes tumor and cell line sample IDs. Found at: doi:10.1371/journal.pone.0011288.s009 (1.05 MB TIF) Figure S7 Tumors derived from T47D but not from MDA-MB- 231 display SABG (+) senescent cells. Two of four T47D tumors PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11288