Macrophage-conditioned medium promotes acquisition of malignant properties in mouse mammary organoids and human ductal structures.Figure 2Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. Representative images of organoids in matrigel/collagen mix. Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. Macrophage-conditioned medium induces invasive protrusions in organoids as compared to control. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages. Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. 3D structure of organoids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). Bi-layered structure of internal luminal cells and external basal myoepithelial cells is established for non-invasive organoids. Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids isolated from ND or HFD mice were cultured in collagen for 2 days. Representative images of organoids cultured in collagen for 2 days. The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape). Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on high fat diet (HFD) (n=3). Organoids isolated from HFD mice were cultured in collagen for 2 days. 3D structure of organoid stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). α-SMA, myoepithelial cell marker, appeared to be loss at invasive protrusions. Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium. Representative images of ductal structures cultured with conditioned medium. Ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Macrophage-conditioned medium induces filling of the duct lumen with cell nuclei compared to control. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.