Figure 4Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of spheroids with invasive protrusions. Error bars represent mean ± SEM from 8 independent experiments (n=2 per condition, at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from mice on ND were cultured in matrigel/collagen mix for 2 days and then stimulated with macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of invasive organoids. 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 M1A - M1 activated, M2A - M2 activated macrophages.Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).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 in presence of amlexanox. Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.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 in presence of amlexanox. Amlexanox inhibits filling of the duct lumen with cell nuclei. 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 M1A - M1 activated, M2 activated macrophages.IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. IKKε CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. IKKε CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.