Fig. 2 | Nature Communications

Fig. 2

From: Macrophages orchestrate breast cancer early dissemination and metastasis

Fig. 2

Intra-epithelial macrophages (IEMs) induce an EMT-like response in early cancer cells. Twenty-week-old MMTV-HER2 mouse mammary glands were stained against E-cadherin and F4/80. E-cadherin localization was analyzed dependent on whether macrophages did not make direct contact to the duct (no M. or distant M.; a) or whether ducts contain IEMs (b). The percentage of individual epithelial cells with disrupted E-cadherin was quantified in four mice and is shown as mean±SEM (c). Statistical analysis: Mann–Whitney test. E-cadherin mRNA expression in whole mammary glands of FVB wild-type (WT, 20wks, N = 4) or 20-week-old MMTV-HER2 (N = 7) mice was determined by qPCR and is shown as mean±SEM by Mann–Whitney test. d Twenty-four-week-old MMTV-HER2 mammary glands (N = 3 per group) were stained against β-catenin and Iba1, a macrophage marker. e, f β-Catenin+ early cancer cells (blue) were more frequent in ducts containing IEMs. g Plots shown as mean±SEM by Mann–Whitney test. The mammary epithelial cell line Comma-1D was grown as a monolayer before the addition of mCherry expressing Raw264.7 macrophages. E-cadherin (h) or β-catenin (i) was stained and the intra-nuclear signal intensity of β-catenin was quantified (j). Plots show nuclear β-catenin signal intensity in individual cells; independent experiments N = 3, Student’s t test. k, l Conditioned medium was harvested from primary mammospheres of 20–22-week-old WT or pre-malignant MMTV-HER2 mice and added to Raw264.7 macrophages or mammary tissue macrophages (MTMs) isolated from pre-malignant MMTV-HER2 mammary glands. Wnt-1 expression is depicted as mean±SEM of three technical replicates. Statistical significance was determined by Student’s t test with 95% confidence interval; individual experiments N = 3 for Raw264.7 and N = 2 for MTMs. Comma-1D cells were grown as monolayers (m) and Raw264.7-mCherry macrophages were added (n) and additionally treated with DKK1 (o). E-cadherin signal intensity in whole section was quantified and is shown as mean±SEM, where each dot represents one microscopic field; independent experiments N = 2, Student’s t test. The pixel intensity of the cells in (n) is background pixel signal for the green channel, ~1000 in pixel intensity

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