Growth of bovine epithelial mammospheres is suppressed by adipose-conditioned medium and rescued by FGF supplementation.

A: Mammospheres formed by bMECs cultured for 3 days within Matrigel are smaller when supplemented with adipose-conditioned medium as compared to bMEC-conditioned or fresh medium. Columns represent average size ± SEM of 28 mammospheres measured in each group. Results of one representative experiment out of three biological replicates are presented. Inset: ammonia levels accumulated in adipose-conditioned, bMEC-conditioned and fresh media. Columns represent average size ± SEM from four analyses of each medium. **Statistically significant difference (p ≤ 0.01) compared to other columns. B: Light microscopy demonstrating the effect of the culture medium on the size of the developing mammospheres. Bar = 50 µm. C: The effect of the culture medium (fold change) on the size of 3-day-developed mammospheres. Columns represent average size ± SEM of 60 mammospheres measured in each group. Different letters above the columns represent statistically significant differences (p ≤ 0.05). D: Immunofluorescent staining of PCNA in representative mammospheres formed by bMECs in fresh and conditioned media. PCNA-positive cells are encircled by a white dotted line. Bar = 50 µm. E: Light microscopy demonstrating no effect of adipose-conditioned medium on the size of mammospheres formed by mMECs. Absence of fibroblasts in cultures supplemented with adipose-conditioned medium is noted. Bar = 100 µm. F: FGF7 and FGF10 supplementation to the adipose-conditioned medium induces mammosphere size increase in a dose-dependent manner. Columns represent average size ± SEM of 60 mammospheres measured in each group. Different letters above the columns represent statistically significant differences (p ≤ 0.05).