Figure 3

Breast cancer stem cell-like properties of OCT4-transduced breast cells (OTBCs) in vitro. (a) Left: Formation of terminal ductal lobular units in three-dimensional (3D) cultures in the presence of Matrigel™ and prolactin. OTBCs were seeded in a 3D culture; 3 weeks later, primary, secondary, and tertiary branching structures were visible. Right: Immunostaining of 3D structures with anti-CK19 (luminal, green) and anti-CK14 (myoepithelial, red) antibodies. (b) Immunostaining of myoepithelial and luminal markers in OTBCs86-L1 cells. Cells were stained for OCT4 (green) and CK14, SMA, Maspin, or CK19 (red) markers. (c) Flow cytometry analysis of antigenic phenotypes characteristic of bi-potent mammary stem cells (CD133^lowCD49f⁺EpCAM^-) and cancer stem cells (CD44⁺CD24^-) in the parental line p86 and the clone OTBCs86-L1. The forward scatter channel was plotted in the y-axis, and the fluorescence of the cell surface antigens was plotted in the x-axis. Virtually identical flow cytometry results were obtained from all analyzed clones (Figure S2 in Additional file 5). Experiments were repeated at least three times with similar results. Data shown are representative of one experiment. EpCAM, epithelial cell adhesion molecule.