Figure 1

Combinatorial signaling of EGF, FGF2 and FGF10 are required for mammary organoid maintenance. (a) Experimental schematic showing mammary glands were surgically dissected and dissociated into single cells. Organoids were grown from single cells and cultured in 3D in the presence of EGF or FGF2 or FGF10 recombinant proteins alone or in a cocktail of all growth factors. The mammary organoids were further analyzed for cell proliferation, expression of mammary lineage markers and transcription factors, and the ability to undergo in vivo organ reconstitution. (b) Representative images showing the size of organoids cultured in a combination of growth factors [All] as compared to the addition of individual growth factors in the basal organoid media at day 10 (Scale bar = 1000 µm). (c) Quantification showing the growth of organoids over a time course starting at day 1 to day 10 (n > 50 organoids were counted per time-point per condition). (d) Organoid proliferation is maintained when cultured in a cocktail of growth factors including EGF, FGF2 and FGF10. Results are expressed as mean ± SD where each dot represents a biological replicate (n = 2) and values were averaged and normalized from three technical replicates. One way ANOVA multiple comparison test was used for computing statistical significance (***p < 0.001). (e) Representative images showing expression of myoepithelial cell markers—Smooth Muscle Actin (SMA, Cyan) in organoids grown under different growth factor condition (Scale bar = 100 µm). (f) Immunofluorescent staining showing the maintenance of Cytokeratin 8/18 (Magenta, Luminal epithelial cell markers) and Cytokeratin 5 (Yellow, basal epithelial cell marker) (Scale bar = 100 µm). The pie chart represents the proportion of K8/18⁺K5⁺, K8/18⁺K5⁻ and K8/18⁻K5⁺ organoids present in each condition. Statistical significance is calculated by Fisher’s Exact test (Two-tailed p value < 0.0001 compared to “All growth factors” condition).