Senescence in Breast Cancer Figure 5. Senescent cell progenitor and immortal cell progenitor subtypes greatly differed in their ability to differentiate into luminal and myoepithelial lineage cells. Senescent cell progenitor and immortal cell progenitor subtype cell lines were studied by immunoperoxidase staining using CD44 and markers for luminal epithelial (CD24, CK19, ER) and myoepithelial (ASMA) lineages. Insets: magnified views of positive cells. Both subtypes have CD44+ cells. Senescent cell progenitor cell lines produced both progenitor-like (CD44+; CD242), as well as ER+ luminal-like and ASMA+ myoepithelial-like cells (except ZR-75-1 for myoepithelial-like cells). Immortal cell progenitor cell lines were defective for generation of ER+ luminal-like or ASMA+ myoepithelial-like cells. Moreover, five of seven cell lines could not generate CD44- cells. The expression of CD24 and CK19 markers did not differ significantly between the two subgroups, except that some immortal cell progenitor subtype cell lines did not express CD24 or CK19. doi:10.1371/journal.pone.0011288.g005 a pattern similar to that of SCP cell lines. A comparison of the two subtypes indicated that the ability to generate both CD44+ and CD44- progeny was significantly associated with the SCP phenotype (P = 0.0046). All five SCP cell lines displayed heterogeneous, but mostly positive ER immunostaining, whereas all seven ICP cell lines never generated ER+ cells. The expression of ER was also significantly associated with the SCP subtype (P = 0.0012), as well as the ability to produce ASMA+ progeny (P = 0.0046). The ICP cell lines did not generate ASMA+ cells, while four out of five SCP cell lines generated rare ASMA+ cells under low-density clonogenic conditions. Interestingly, the abun- dance of ASMA+ cells was much higher in the two SCP cell lines that were tested at high cell density (Fig. S5). This suggests that either the production of ASMA+ cells is enhanced at high cell density, or these myoepithelial-like cells display limited survival under long-term culture conditions. We did not find a strong association between the expression of CD24 and CK19 markers and cell subtype. All five SCP cell lines and three ICP cell lines PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11288