Figure 5

Endothelial-derived effects are mediated trough soluble factors. A) Schematic figure of a Transwell co-culture assay. BRENCs (grey) were seeded on Transwell (TW) filter inserts and allowed to reach confluency. Epithelial cells (orange) were embedded into Matrigel and seeded in the lower chamber (a). Hematoxylin stained BRENCs on a TW insert (b). Bar = 200 μm. B) Phase contrast images of controls and co-cultures in rBM. BRENCs were able to stimulate proliferation of MCF10a (a, b) and D382 (c, d) when separated by a Transwell filter. BRENCs were also able to stimulate proliferation of MCF7 (e, f), T47-D (g, h) and MDA-MB-231 (i, j) when separated by a Transwell filter. Bar = 50 μm. C) Epithelial colony size is increased in co-culture with BRENCs. A significant (P < 0.0001) increase in colony size of the cell lines MCF10a, D382 and MCF7 was seen in co-cultures (black bars) compared to controls (white bars). No significant proliferative effect was seen in T47-D and MDA-MB-231 co-culture with BRENCs. D) Cloning efficiency of normal breast epithelial cells is enhanced in co-culture with BRENCs. Colony formation was increased for the non-malignant cell lines only (MCF10a and D382). In contrast, similar colony count was seen in controls and co-cultures for the cancer lines.