Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
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The RNA-seq data that support the findings of this study have been deposited in the GEO under accession code GSE119869. Previously published microarray data that were re-analysed here are available under accession code GSE56755 (ref. 48). All other data supporting the findings of this study are available from the corresponding author on reasonable request.
FIJI macros are available from the authors on request.
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We thank W. Alexander, M. Kauppi and A. Stock for their assistance with the chimaera experiments; F. Jackling for animal management; M. Chopin and S. Nutt for providing mice; Y. Hu for assistance with bioinformatics; J. Whittle, S. Naik, G. Belz, S. Heinzel, J. Schreuder and D. Lin for discussions; and C. Nowell at the MIPS imaging facility. We are grateful to the WEHI Centre for Dynamic Imaging, flow cytometry and animal facilities. This work was supported by the Australian National Health and Medical Research Council (NHMRC) grant nos 1016701, 1054618, 1100807 and 1113133; NHMRC IRIISS; the Victorian State Government through VCA funding and Operational Infrastructure Support and the Australian Cancer Research Foundation. C.A.D. was supported by an Australian Government Research Training Program Scholarship. A.C.R. was supported by a National Breast Cancer Foundation (NBCF)/Cure Cancer Australia Fellowship. S.N.M., G.K.S., G.J.L. and J.E.V. were supported by NHMRC Fellowships (grant nos 1136550, 1058892, 1078730, and 1037230 and 1102742, respectively).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Macrophage and dendritic cell populations in the mammary gland (supporting information for Fig. 1).
a, Analysis of a whole-mount 3D confocal image of mammary tissue from a ten-week-old CD11c-GFP mouse immunostained for GFP and keratin 5 (K5). K5 surface (magenta), GFP signal spots (cyan) (left). Image representative of 2 mice. Scale bar, 200 µm. Middle panel: distance from spot centre to K5 surface. x-axis: centre of 10 µm bins. Right panel, box blot: minimum, maximum and quartiles. n=663 cells (>5 µm) and 591 cells (<5 µm) pooled from 2 mice. Normalised CD11b mean intensity within GFP spots (diameter 10 µm). b, Overlay of MØ1/2, MØ3 and CD45+ cells by FACS (gated as in Fig. 1c). c, FACS quantification of nine-week-old mice treated with 400 µg anti-Csf1r or isotype by i.p. injection 7, 4 and 2 days before collection (n=3 mice). DC2, CD11b+ DCs; DC1, CD11b−CD24hi DCs. Scale bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0231 (MØ1), *P=0.0159 (MØ2), *P=0.0101 (MØ3), ns P=0.6122 (DC1), ns P=0.8956 (DC2). d, FACS plots from mice at 4, 6 and 20 weeks of age (n=3 mice, 4, 6 weeks; n=4 mice, 20 weeks). Same samples shown in Fig. 3g. e, FACS plots from mice at 9 weeks (n=2 mice). f, FACS plots from mice at 9 weeks and Lyve1-FITC mean MFI normalised to autofluorescence (n=2 mice). g, YFP MFI in Irf8-YFP mice normalised to autofluorescence. DC1, CD11b− DCs; DC2, CD11b+ DCs (n=2 mice). h, 3D image of Irf8-YFP tissue at 16 weeks immunostained for K5 (magenta) and YFP (cyan). Image representative of 2 mice. Inset: View along duct axis. Arrows indicate Irf8hi cells on outer basal surface. Scale bars, 100 µm (overview) and 30 µm (enlargement). i, Analysis of (h): distance from YFP+ spot centre to K5 surface (n=2 mice). j, Analysis from a 3D image of Csf1r-GFP tissue immunostained for GFP and K5 (Fig. 1d). Histogram of distance from GFP+ spot centre to K5 surface (n=2 mice). Source data
a, Quantification of MØs and DCs by FACS in six-week-old CD11c-CreT/+Irf8fl/fl and CD11c-Cre+/+Irf8fl/fl mice. Fold-change % CD45+ cells was calculated relative to control (n=3 mice, control; n=4, knockout). Mean values are displayed. Error bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0142, ns: P=0.6517 (MØ3), P=0.9552 (MØ1/2), P=0.8773 (DC2). b, Carmine-stained whole-mounts from six-week-old CD11c-CreT/+Irf8fl/fl and CD11c-Cre+/+Irf8fl/fl mice and branch quantification (n=6 mice, control; n=11, knockout). Error bars, s.e.m. Two-tailed Student’s t-test. Scale bars, 2 mm. c, FACS analysis one day after DT treatment of CD11b-DTR mice (n=3 mice, control; n=4 mice, depletion). Eos: eosinophil, Neut: neutrophil, Mono: monocyte. Means are stated. Error bars, s.e.m. Two-way ANOVA with Sidak correction ****P<0.0001. d, Whole-mounts from CD11b-DTR or WT mice treated with DT (5 weeks) and collected at 6 weeks (n=4 mice, control; n=5 mice, depletion). Error bars, s.e.m. Two-tailed Student’s t-test **P=0.0018. Scale bars, 2 mm. e, FACS analysis of CD11c-CreT/+RBPJfl/fl (RBPJ cKO) and CD11c-Cre+/+RBPJfl/fl (control) mice at 6 weeks, frequency relative to epithelial cells (CD45−CD24+) (Two-way ANOVA with Sidak correction, ns: all P>0.9999) and MFI relative to control mean. Two-way ANOVA with Sidak correction ****P<0.0001, ns: P=0.5882 (MHCII), n=0.1659 (CD11c), n=0.9271 (Ly6C) (n=5 mice, controls; n=3 mice, cKO). Error bars, s.e.m. f, Whole-mounts from control and RBPJ cKO mice and quantification of ductal area and branch frequency (n=5 mice, control; n=3 mice, cKO). Error bars, s.e.m. Two-sided Student’s t-tests, P=0.6548 (area), P=0.0538 (branches). Scale bars, 2 mm. g, 3D images from control and RBPJ cKO mice immunostained for MHCII (yellow) and K5 (magenta). Opaque signal rendering as in Fig. 3c. Image representative of 5 mice (control) and 3 mice (cKO). Scale bars, 50 µm. Source data
a, Top 50 positive SM signature genes. Heatmap shows mean-centred log2-expression with genes ranked by average FDR across pairwise comparisons (see Methods for a–i) (n=2 samples per population, each pooled from 12 mice, applies to a–f). b, Top 50 positive DC1 signature genes. c, Top 50 positive DC2 signature genes. d, DE genes between DMs versus SMs. Left heatmap shows most significant genes upregulated in DMs, right shows most significant down-regulated genes. e, Heat map showing relative expression of genes previously associated with MØs4 in the mammary DC/MØ populations. f, Heat map showing relative expression of genes previously associated with DCs32 in the mammary DC/MØ populations. g, GFP MFI from FACS analysis of Cx3cr1GFP/+ mice relative to wild-type (WT) controls (Fig. 2f) (n=2 mice). h, Barcode enrichment plots showing that genes associated with MHCIIloLyve1hi lung MØs26 are enriched in mammary MØ1/2 (roast p = 4e-5) whereas genes associated with MHCIIhiLyve1lo lung MØs26 are enriched in DMs (roast p = 5e-5). Genes are ordered in the plot right to left from most up- to most down-regulated in DMs versus SMs (Fig. 2). The x axis shows moderated t-statistics. Vertical bars designate MHCIIloLyve1hi or MHCIIhiLyve1lo lung MØ genes and the worms show relative enrichment. i, Epithelial and MØ expression of known receptor-ligand interactions for genes specifically associated with DMs or MØs. Epithelial gene expression data are from Sheridan et al., 201565. Lum, luminal; Prog, progenitor. Source data
Extended Data Fig. 4 Intra-epithelial macrophages are abundant within the mammary epithelium throughout postnatal development.
a, Optical sections of mammary ducts at 9 weeks of age, immunostained for K5 (magenta), MHCII (yellow) and GFP, YFP or CD11b (cyan) in WT or indicated reporter mice. Arrows indicate intra-epithelial MØs. Hollow arrowhead indicates a CD11b+ stromal cell. Images representative of 3 mice (Csf1r, CD11c and CD11b) and 6 mice (Cx3cr1). Scale bars, 20 μm. b, 3D image of a mammary gland at 2 weeks of age, immunostained for K5 (magenta) and MHCII (yellow). Enlargement: inner duct surface with opaque signal. Image representative of 3 mice. Scale bars, 200 µm (overview) and 20 µm (enlargement). c, 3D image of terminal end buds (TEBs) at 5 weeks and enlarged optical section, immunostained for GFP (cyan) and K5 (magenta) and labelled for F-actin (yellow). Image representative of 3 mice. Arrows indicate dendritic-shaped Cx3cr1hi cells within the TEB. Scale bars, 100 µm (overview) and 40 µm (enlargement). d, 3D images of ducts of the nipple, mid and distal regions of glands and enlarged optical sections, from a Cx3cr1GFP/+ mouse at 8 weeks, immunostained for K5 (magenta), GFP (cyan) and keratin 8 (K8, yellow). Images representative of 2 mice. Arrows indicate Cx3cr1hi DMs between the K8+ luminal and K5+ basal layers. Scale bars, 50 µm.
a, 3D image of a Cx3cr1GFP/+ mammary rudiment at E18.5 with enlarged optical sections of the distal (i) and nipple (ii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate Cx3cr1+MHCII− cells. b, 3D image of a Cx3cr1GFP/+ mammary gland at postnatal day (P) 4 with enlarged optical sections of the distal (i), mid (ii) and nipple (iii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate Cx3cr1+MHCII– cells. c, 3D image of an entire Cx3cr1GFP/+ mammary gland at P7 with enlarged optical sections of the distal (i), mid (ii) and nipple (iii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate dendritic Cx3cr1+MHCIIlo/+ cells. All scale bars, 300 µm (overview) and 100 µm (enlargements).
a, Photo of a mouse prepared for intravital imaging (left) and the exposed mammary gland after intraductal injection of fluorescent beads (right). b, Isolation of DM MHCII signal in 3D-IVM images from Elf5-GFP mice with labelling by anti-MHCII AF647 antibody (see Methods, 6 experiments). Left: raw GFP (magenta) and MHCII (cyan) signal. Middle: addition of GFP surface rendering (white). Right: GFP and masked duct-adjacent DM MHCII signal (yellow). Scale bar, 30 µm. c, Time-points from a 3D-IVM movie (Supplementary Video 3) in which precise laser damage was induced in the epithelium of a Cx3cr1GFP/+ mouse at 9 weeks. Images representative of 3 mice. Time hrs:mins. Scale bar, 20 µm. d, 3D-IVM of Elf5-GFP mammary tissue at 3 days involution (Supplementary Video 5) showing GFP (magenta) and anti-MHCII AF647 antibody (yellow). Left: overview. Right: enlarged 3D projections at time-points throughout phagocytosis and the outlined volume viewed from the side. Arrows indicate GFP+ cells within DMs. Images acquired every 5 mins. Images representative of 3 mice. Scale bars, 100 µm (left), 20 µm (enlargements).
a, MØ FACS profiles throughout postnatal development and DM/epithelial cell (CD45–CD24+) ratio in virgin and pregnant glands. n=3, 5 weeks, preg d12.5 and preg d16.5 ; n=5, 9 weeks; n=6, lactation. Error bars, s.e.m. b, 3D images of 11 week-old adult and 16.5 d pregnant glands and enlarged optical sections, immunostained for K5 (magenta) and MHCII (yellow) and labelled for EdU (cyan). Mice were treated with EdU 2 hrs prior to collection. Images representative of 4 mice. Hollow arrow-head indicates an EdU– DM. Arrow indicates an EdU+ DM. Scale bars, 100 µm (overviews) and 15 µm (enlargements). c, 3D image and enlarged optical section of Cx3cr1GFP/+ tissue at 4 days involution, immunostained for GFP and labelled for F-actin. Images representative of 2 mice. Dotted lines indicate the outer edge of the F-actinhi basal layer. Scale bar, 30 µm. d, Optical section from a 3D image of Elf5-GFP tissue at 3 days involution. Image representative of 3 mice. Arrows indicate large GFPlo alveolar cells surrounded by MHCII signal. Hollow arrow-heads indicate binucleated cells. Scale bar, 20 µm. W, weeks; d, days; lact, lactation. Source data
a, FACS of CD11c-DTR mice at 1 day involution, treated with DT or PBS upon forced weaning at 14 d lactation (n=2 mice). b, MØ and DC frequency at 4 d involution by FACS after treatment as in a (n=4 mice, CD11c-DTR; n=10, control). Percent CD45+ cells normalized to controls. Control: CD11c-DTR mice treated with PBS or WT mice treated with DT. DC1, CD11bloCD24hi DCs; DC2, CD11b+CD24int DCs. Error bars, s.e.m. Two-way ANOVA with Sidak correction. ***P=0.0007, ****P<0.0001, ns P>0.9999 (Ly6C+CD11c– MØs), P=0.0941 (DC2). c, Optical sections of CD11c-DTR tissue at 4 d involution after treatment as in (a). Images representative of 3 mice (PBS) and 4 mice (DT). Immunostaining for CC3 (cyan) and MHCII (yellow) and labelling for F-actin (magenta). Scale bars, 100 µm. d, FACS quantification at 4 d involution after treatment with AFS98 anti-Csf1r antibody or isotype control one day prior to weaning, at weaning and at 1 d involution (n=3 mice). Fold-change percent total cells relative to control mean. Monocytes CD64+F4/80+Ly6ChiMHCII–. Error bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0212, ***P=0.0008, ns: see Source data. e, H&E staining of tissue from d with alveolar lumen area outlined and quantified (n=3 mice). Error bars, s.e.m. Two-sided Student’s t-test *P=0.0261. Scale bars, 100 µm. f, 3D images of tissue from d immunostained for MHCII (yellow) and CC3 (cyan) and labelled for F-actin (magenta). Images representative of 3 mice. Scale bars, 20 µm. g, Optical sections from a 3D image of Cx3cr1GFP/+ tissue at 6 d involution, immunostained for GFP (yellow) and CC3 (cyan) and labelled for F-actin (magenta). Images representative of 2 mice. Arrows indicate CC3+ DMs. Scale bars, 15 µm. Source data
a, FACS plots from an MMTV-PyMT tumour showing gating strategy (n=4 mice) b, FACS plots of MØs (CD45+Ly6G–CD64+CD24lo) in age-matched WT FVB/N mammary glands, tumours and tumour-adjacent hyperplastic tissue from MMTV-PyMT, MMTV-Wnt1 and MMTV-Neu mice (n=4 mice per model). c, Quantification of myeloid cell frequencies by FACS in pooled WT controls, tumours and hyperplastic tissue. Eos: eosinophil, Neut: neutrophil, Mono: monocyte. Values are averages (n=6 mice, WT; n=4 mice, others). d, Quantification of MØ subsets throughout tumorigenesis in MMTV-PyMT/Cx3cr1GFP/+ mice, corresponding to Fig. 7b (n=2 mice). e, Immunostaining of MMTV-PyMT tumour tissue for TMEM119. Images representative of 2 mice. Control panel shown below. Scale bar, 10 µm. Source data
Whole-mount 3D confocal imaging of DMs in lactation. An animation of a whole-mount 3D confocal image of mammary tissue at 14 d lactation (Fig. 3d), immunostained for K5 (Magenta) and MHCII (yellow), and labelled for F-actin (pink).
Intravital imaging of steady-state DM behaviour. An animation of 3D-IVM of mammary ducts in an Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Fig. 4a–c). The movie cycles over a 6-h time span with images acquired every 10 min. GFP, magenta; masked DM MHCII, yellow (see Extended Data Fig. 6b and Methods); and stromal MHCII, cyan. Time in h:min (n = 6 mice).
Intravital imaging of DM response to epithelial damage. An animation of 3D-IVM of a mammary duct in a Cx3cr1GFP/+ mouse (Extended Data Fig. 6c). The movie cycles through time points prior to damage showing the arrangement of GFPhi DMs (yellow) around a duct, then views an optical section through DMs before and after precise multiphoton laser damage at 4 h (bolt symbol). Images were acquired every 3 min (n = 3 mice).
Intravital imaging of DM response to apoptosis. 3D-IVM of a mammary duct in a nine-week-old Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Fig. 4e). GFP, magenta; Masked DM MHCII, yellow. Apoptotic cells are labelled with PI (white). Images were acquired every 5 min (n =3 mice).
Intravital imaging of DM behaviour during involution. 3D-IVM of alveoli at 3 d involution in an Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Extended Data Fig. 6d). GFP, magenta; MHCII, yellow. Images were acquired every 5 min (n = 3 mice).
DM-like TAMs form a dendritic network within mammary tumours. An animation of a whole-mount 3D confocal image of MMTV–PyMT/Cx3cr1GFP/+ tumour tissue immunostained for GFP (yellow) and labelled with DAPI (white; Fig. 7c; n = 4 mice).
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Dawson, C.A., Pal, B., Vaillant, F. et al. Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling. Nat Cell Biol 22, 546–558 (2020). https://doi.org/10.1038/s41556-020-0505-0
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