generated heterogeneously staining colonies for CD24 expression. Similarly, all five SCP cell lines, as well as three ICP cell lines, expressed CK19, but homogenously. Typical features of senescent progenitor and immortal progenitor breast cancer cell lines As summarized in Fig. 6, SCP and ICP subtype cell lines displayed several subtype-specific features. All SCP cell lines produced differentiated and senescent cells, in addition to putative CD44+/CD242 stem/progenitor cells. Indeed, all of them produced ER+ and CD24+ luminal-like cells and most of them (n = 4/5) also produced ASMA+ myoepithelial-like cells. In contrast, five of the seven ICP cell lines never produced CD44- cells, suggesting they cannot generate differentiated progeny under the experimental conditions tested. In confirmation of this hypothesis, four ICP cell lines only produced CD44+/CD242/ ER-/CK19-/ASMA- stem/progenitor-like, but never differentiat- ed cells. Furthermore, all seven ICP cell lines were unable to produce ASMA+ myoepithelial-like, ER+ luminal-like or SABG+ senescent cells. CD24 and CD19 luminal lineage markers were expressed in three cell lines, one of which was fully positive for the CD44 stem/progenitor marker. SCP and ICP subtype cell lines correlate with distinct breast tumor subtypes Distinct cell-type features associated with SCP and ICP subtypes suggest that they may be phenocopies of molecularly defined Figure 6. Typical features of senescent progenitor and immortal progenitor breast cancer cell lines. doi:10.1371/journal.pone.0011288.g006 Senescence in Breast Cancer breast tumor subtypes [1,2,3,30]. As the prognosis and therapeutic response of each subtype is different [2,3], we questioned whether we could assign SCP and ICP cell lines to known molecular subtypes of breast tumors. Using cell line and primary tumor gene expression datasets, we conducted a hierarchical clustering analysis. The ‘‘intrinsic gene set’’ data generated by Sorlie et al. [2] to classify breast tumors into five molecular subtypes was used to filter cell line data generated by Charafe-Jauffret et al. [13]. A set of 175 genes was common between the two data sets. Sixty- eight tumors and 31 cell lines were subjected to pair-wise complete-linkage hierarchical clustering and distance measure- ments. This tumor–cell line combined analysis produced two major clusters. One cluster was composed of basal and luminal B subtype tumors and five of six ICP cell lines. The other cluster included luminal A, ERBB2+, and ‘‘normal-like’’ subtype tumors and all five SCP subtype cells. Four cell lines clustered with the luminal A tumor subclass. Finally. one cell line clustered with the ‘‘normal-like’’ subclass (Fig. 7A). A full list of clustered tumors and cell lines is provided in Fig. S6. Luminal A tumors clustering with our SCP subtype cell lines displayed the longest tumor-free, distant metastasis-free, and overall survival rates. In contrast, basal and luminal B tumors clustering with our ICP subtype cell lines had the worst prognosis, with shorter tumor-free, distant metastasis-free, and overall survival times [2]. Our cluster analysis suggested that the ability to generate differentiated and senescent progeny characterized breast cancers with poor tumorigenicity, and that resistance to differentiation and senescence was indicative of more aggressive tumorigenicity. We compared in vivo intrinsic tumorigenic behaviors of the SCP cell line T47D and the ICP cell line MDA-MB-231 in female (n = 5 for each cell line) and male (n = 4 for each cell line) CD1 nude mice Fig. 7B). MDA-MB-231 cells displayed higher tumorigenicity than T47D cells. In total, we observed progressively growing tumors in five of nine animals injected with MDA-MB-231. In contrast, T47D formed smaller and regressing tumors in nine of nine animals (P,0.03). Interestingly, the difference in tumorigenicity between these two cell lines was more pronounced in male animals (P,0.05 after 20 days post-injection). To test whether the difference in tumorige- nicity between MDA-MB-231 and T47D cells was correlated with spontaneous in vivo senescence, we analyzed four tumors from each cell line. Two T47D tumors displayed positive SABG staining, whereas no positive staining was observed with all four MDA-MB-231 tumors (Fig. S7). Discussion In recent years, phenotypic heterogeneity of breast cancers has been correlated with genetic and molecular heterogeneity [1,3]. Breast cancer subtypes may represent cancers originating from different progenitor cells. Molecular and phenotypic heterogeneity and associated clinical manifestations of breast tumor subtypes have been related to the type of hypothetical tumor progenitor cells originating from a hypothetical mammary epithelial stem cell or from downstream progenitor cells [4,5]. This hypothesis has not been fully validated, mainly because a hierarchical map of cells involved in mammary epitheliogenesis has not yet been established. To better understand phenotypic differences between breast cancer subtypes, we applied senescence as a surrogate marker for the potential to generate terminally differentiated progeny. We completed these studies with markers for breast stem/progenitor and differentiated luminal and myoepithelial lineage cells. The use of low-density clonogenic conditions allowed us to follow the fate PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11288