Figure 3

Pure luminobasal (pLB) proliferation is suppressed by pure luminal (pLUM) cells via a diffusible factor. pLUM, pLB and ratios of pLUM:pLB cell mixtures were seeded onto solidified growth factor (GF)-reduced Matrigel. Three-dimensional colonies were cultured for 7 days in hormone-free conditions. Bromodeoxyuridine (BrdU) was added, Matrigel blocks were sandwiched between HistoGel, paraffin-embedded and sectioned for immunohistochemistry (IHC). (A) Sections dual stained for CK5 (red) and CK8/18 or BrdU (green), counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bars equal 20 μM. (B) Quantitation of BrdU incorporation into pLB or pLUM cells in pure cells (set at 100%) or mixed-cell colonies. (C) Same as A and B, but MCF-7 were substituted for pLUM. (D) Media-conditioned (CM) by no cells (control), or by pLB, pLUM or MCF7 cells growing in 2D on plastic, were collected. Separately, pLB, pLUM or MCF-7 cells were grown as three-dimensional Matrigel colonies in regular media for 48 h, then switched to the CM media shown for 48 h. Colonies were photographed and their diameters in CM were compared to colony diameters in control media; *P ≤0.05; **P ≤0.01; ***P ≤0.001; ****P ≤0.0001.