Figure 3

BRENCs induce clonal growth of normal and malignant breast epithelial cell lines. A) BRENCs generate positive environment for clonal growth of breast epithelial cells. MCF10A, D382, MCF7, T47-D and MDA-MB-231 cells were seeded at a clonal dilution, alone or in co-culture with BRENCs. When cultured alone there was very limited colony formation, most seeded cells stayed as non-proliferative single cells (a, c, e, g, i). In contrast, co-cultures of BRENCs with all cell lines resulted in dramatic increase in colony growth. MCF10A cells formed multiacinar-like structures (b), whereas D382 formed solid round colonies (d). both MCF7 and T47-D formed solid round colonies (f, h). MDA-MB-231 formed mesenchymal like colonies (j). Bar = 100 μm. B) Immunophenotypic characterization of endothelial-epithelial co-cultures. β4-integrin staining reveals the different phenotype of MCF10A and MDA-MB-231, where MCF10A forms dense multiacinar structures delineated by β4-integrin expression but MDA-MB-231 forms colonies of loosely connected cells with diffused staining pattern of β4-integrin. (a-b). Note the expression pattern of β4-integrin. CD31 staining shows the distribution of BRENCs around the colonies (c-d). ki67 expression levels are similar in both cell lines (d-e) and minimal expression of cleaved caspase 3 is seen (g-h) Bar = a-d 100 μm and e-h = 50 μm. C) Colony size is increased in co-cultures with BRENCs. Colony size (n > 100 for all cell lines) of epithelial cells co-cultured with BRENCs increased significantly compared with controls (P < 0.0001 for all co-cultures). MCF10A gave the biggest response, with a 15.5 fold increase in colony size, averaging at 320.6 μm diameter. Colony size varied greatly between cell lines. D) Colony growth over time. Most cell lines followed a linear growth pattern. Note however, the drastic change in growth of MDA-MB-231 cells after day 9 (light grey line).