Fig. 6 | Nature Communications

Fig. 6

From: Macrophages orchestrate breast cancer early dissemination and metastasis

Fig. 6

HER2 activates NF-κB and upregulates CCL2. a Western blot for NF-κB subunit phospho-p65 in mammosphere (MS) lysates from 20-week-old MMTV-HER2 mice. One representative blot of three independent experiments is shown. b CCL2 expression in MS from FVB wild-type and pre-malignant MMTV-HER2 mammary glands (MGs). Technical replicates, three experiments (3 mice/group); statistical analysis: with 95% confidence interval by Mann–Whitney test. c MGs from FVB wild-type and 22-week-old MMTV-HER2 mice stained against CCL2, HER2, and CCR2. Bars: 25 μm. Inset, CCR2 signal, zoom factor 1x; full image in Supplementary Fig. 5i. CCL2 intensity was quantified using ROI tool in Metamorph (Supplementary Fig. 5h). d ELISA of CCL2 in 24 h conditioned media of WT or HER2+ MS isolated from two animals. P value with 95% confidence interval by Mann–Whitney test—SEM shown. eg CCL2 staining in acini cultures from MMTV-HER2 MGs (20–24 weeks (wks)) grown for 5 days and then DMSO-treated (vehicle control; e), 1 μM lapatinib (f) or 1 μM IKKi compound A (g) for 24 h. Bar: 25 μm. hj MG acini treated with DMSO (vehicle), lapatinib (1 μM), IKKi compound A (1 μM), or CCR2 inhibitor RS504393 (1 μM). Primary MG macrophages were then added and the percentage of acini associated with or without macrophages (h) was quantified (i; bar: 25 μm; zoom factor 2.6x); j mean±SEM; each dot one technical replicate; two independent experiments; statistical analysis: Mann–Whitney test—SEM shown). km Twenty-week-old MMTV-HER2 mice were treated with CCR2 inhibitior RS504393 (2 mg/kg i.p. daily) for 2 weeks and stained against E-cadherin and F4/80 (k, l; bars: 25 μm). m Intra-epithelial macrophage (IEM) containing ducts were quantified (with 95% confidence interval by Mann–Whitney test; each dot: mean±SEM of one animal). n Quantification of circulating cancer cells (CCC) in the mice in the experiment in km (statistical analysis: Mann–Whitney test—SEM shown). CCC quantification was performed as in Fig. 3h. o Twenty-week-old MMTV-HER2 mice were treated locally with 1 mg/kg CCR2 inhibitor injected into the MG fat pad every day for 5 days and vehicle control into the contra-lateral gland. Glands were stained against F4/80 and IEM containing ducts were quantified (o: 5 mice per group; statistical analysis: Mann–Whitney test—SEM shown). Values in o normalized to the IEM content of each contra-lateral control-treated gland