Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway (2019)
Figure 4

Fig 4: The macrophage-mediated transforming effect is reduced by amlexanox, an inhibitor of IKKε/TBK1, or upon IKKε deletion.

A) Amlexanox 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). All the data shown without the use of amlexanox are also included in Fig. 1A.

B-E) 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.

B) 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.

C,D) Amlexanox reduces filling of the spheroid lumen with cell nuclei.

C) Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled).

D) Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids).

C,D) 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. All the data shown without the use of amlexanox are also included in Fig. EV3D and 1C respectively.

E) 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). All the data shown without the use of amlexanox are also included in Fig. 1I.

F-H) Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3).

F) 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). All the data shown without the use of amlexanox are also included in Fig. 2B.

G,H) 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). All the data shown without the use of amlexanox are also included in Fig. 2E.

I,J) 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.

I) 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.

J) 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. All the data shown without the use of amlexanox are also included in Fig. 2H.

K,L) IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line.

K) 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.

L) 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.

K,L) 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 D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages. Aml. - amlexanox (50 μM). *p < 0.05 as measured by two-tailed students t-test (exact P values are shown in Table EV3).