Effect of fibroblasts on co-unit structure. Incorporation of normal breast fibroblasts into myoepithelial/MCF-7 cultures. Non pre-aggregated cells were mixed and cultured in a collagen gel for up to one week to establish if fibroblasts influence epithelial co-unit formation. (a) (a)i,ii: At day 1, MCF-7 cells, myoepithelial cells (cell trackered red) and fibroblasts (cell trackered green) were intermingled. (a)iii,iv: By day 3, MCF-7 clusters were beginning to form and (a)v,vi these were more prominent by day 5. (a)vii,viii: Co-units were formed by day 7 with normal fibroblasts located around the epithelial structures (low power image). Incorporation of tumour-associated fibroblast (TAF) into myoepithelial/MCF-7 into 7 day co-cultures. (b) (b)i,ii: The homing of myoepithelial cells to MCF-7 cells is disrupted on addition of TAFs, with the cells reverting to an intermingled mixed population. (b)iii: The effect on the culture is particularly evident at low-power magnification. (c) Quantitation of co-units revealed a decrease from 7.5 to 1.2 in the presence of TAFs compared with normal fibroblasts (p = 0.001). (d) These effects could be replicated in cultures grown for seven days in the presence of conditioned medium from the fibroblast populations with disruption of co-unit architecture (d)ii in the presence of TAF conditioning media but not (d)i normal fibroblast conditioning media.