Research Article

Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche

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Science  29 Jun 2018:
Vol. 360, Issue 6396, eaan4153
DOI: 10.1126/science.aan4153

Cross-talk in the mammary gland

Macrophages engulf damaged and dead cells to clear infection, but they also participate in tissue regeneration. Chakrabarti et al. expand the macrophage repertoire for mammary gland development (see the Perspective by Kannan and Eaves). Mammary gland stem cells secrete the Notch ligand Dll1 and activate Notch signaling, which promotes survival of adjacent macrophages. This stimulates production of Wnt ligands, which signal back to the mammary gland stem cells. This cross-talk plays an important role in coordinating mammary gland development, tissue homeostasis, and, not least, breast cancer.

Science, this issue p. eaan4153; see also p. 1401

Structured Abstract

INTRODUCTION

The stem cell niche plays a crucial role in regulating key stem cell properties, including self-renewal, differentiation, and cell fate change. Although stem cell niches in several organs have been well described, the cellular and molecular characteristics of the mammary gland stem cell (MaSC) niche remain largely underexplored. Stromal cell populations—including fibroblasts, macrophages, and other immune cells—are important for mammary gland development and have been implicated in MaSC niche function. However, the signaling mechanisms driving mammary stromal cell–dependent regulation of MaSC activity remain elusive. Insight into the cross-talk between MaSCs and the niche cells is important for understanding both normal tissue homeostasis and disease conditions such as breast cancer.

RATIONALE

Notch signaling is broadly involved in cell fate regulation during development. Although Notch receptors have been implicated in various aspects of mammary gland development, the role of Notch ligands in MaSC regulation is less clear. In this study, we focus on the Notch ligand Dll1, which is highly expressed in MaSC-enriched mammary epithelial cell (MEC) populations. Conditional knockout (cKO) of Dll1 in MECs resulted in a significant delay in branching morphogenesis during mammary gland development and a deficiency in alveoli formation during pregnancy and lactation, suggesting a key role of Dll1-mediated pathways in mammary gland development.

RESULTS

We found that Dll1cKO mice have a reduced number of MaSCs at different stages of mammary gland development in virgin and pregnant animals. Furthermore, using Dll1 reporter mice, we found that Dll1 expression is enriched in MaSCs, and Dll1+ MaSCs have a greater regenerative potential than Dll1 MaSCs. Lineage tracing with Dll1-Cre-ERT2;dTomato reporter mice revealed that Dll1+ cells can produce both basal and luminal cells. Dll1cKO mice exhibit a significant reduction in the number of mammary gland macrophages. The mammary macrophages have molecular features, including enrichment of Wnt and Notch signaling pathway components, that are distinct from those of macrophages in other tissue. Dll1 binds to Notch2 and Notch3 on mammary macrophages to activate Notch signaling, which is necessary to sustain macrophage numbers in the niche and their MaSC-promoting activity. Using a MaSC-macrophage coculture system, we also showed that MaSC-derived Dll1 induces expression of Wnt ligands—such as Wnt3, Wnt10, and Wnt16—from macrophages, and these ligands feed back to MaSCs to promote their stem cell activities. In vivo experiments involving genetic and pharmacological depletion of macrophages, as well as macrophage-specific deficiency of Notch signaling, further validated the crucial role of mammary macrophages in sustaining MaSC activity.

CONCLUSION

We identified Notch ligand Dll1 as a marker that is enriched in MaSCs and demonstrated that Dll1+ MaSCs can generate both basal and luminal cells. Our study establishes macrophages as important cellular components of the MaSC niche through intercellular coupling of Notch and Wnt signaling. Dll1 produced from MaSCs activates Notch signaling in macrophages to sustain their numbers and enhance the expression of Wnt ligands, which in turn supports Wnt signaling in MaSCs to maintain stem cell activity. Our study defines a Dll1-mediated MaSC niche in which the survival and function of MaSCs and stromal macrophages are mutually regulated by cross-talk between the two cell types through Notch and Wnt signaling.

Dll1+ MaSCs interact with mammary macrophages through Notch and Wnt signaling.

Dll1 conditional knockout (Dll1cKO) mice display delayed ductal growth compared with wild-type (WT) mice (compare lower and upper left). In mouse mammary glands (upper right), F4/80+ macrophages (brown) are in close proximity to basal MECs. In the schematic model (lower right), Dll1 in MaSCs (pink) activates Notch signaling in macrophages, increasing their production of Wnt ligands, which in turn promote MaSC activity.

Abstract

The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. We show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche in mouse models. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feedback loop that promotes the function of Dll1-expressing MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1-mediated Notch signaling.

Mammary epithelial cells (MECs) are composed of two major cell types—basal and luminal cells—both of which derive from mammary gland stem cells (MaSCs) during puberty and each round of pregnancy and lactation (1, 2). The existence of MaSCs has been demonstrated by transplantation (3, 4) and lineage tracing experiments (5, 6). MECs are surrounded by various stromal cell types, including adipocytes, fibroblasts, macrophages, endothelial cells, and lymphoid cells (7). Although some of these cells contribute to mammary gland development and homeostasis (814), little is known about their functional involvement in regulating MaSCs.

Notch and Wnt signaling pathways are key regulators of essential developmental processes in the mammary gland, including stem cell maintenance, cell fate decisions, and dedifferentiation (3, 1520). Notably, studies on Notch signaling in mammary gland development have primarily focused on the receptors (1517, 21, 22), whereas relatively little is known about the role of specific Notch ligands. Similarly, Wnt signaling is well established for sustaining adult stem cells in many organs (23), including MaSCs (3, 1820, 24). Several studies have shown that Wnt ligands such as Wnt3a and Wnt4 are important for the self-renewal of MaSCs (18, 25); however, these Wnt ligands are not expressed by the basal stem cells, which suggests that an adjacent MaSC niche might be responsible for secretion of the ligands. Indeed, recent studies have shown that Wnt4 controls MaSC function through luminal-myoepithelial cross-talk (25). It remains unclear whether stromal niche cells can also produce Wnt ligands to regulate MaSCs.

In this study, we demonstrate that Notch ligand Dll1 expression is enriched in MaSCs and that Dll1 activates Notch signaling in stromal macrophages to induce their expression of Wnt ligands, which feed back to MaSCs to promote stem cell activity. Our study defines a Dll1-mediated MaSC niche in which the survival and function of MaSCs and stromal macrophages is mutually regulated by cross-talk through Notch and Wnt signaling.

Results

Dll1 is required for mammary morphogenesis in virgin and pregnant mammary glands

Our recent gene expression profiling analysis of different populations of mammary gland cells (26) revealed that Dll1 is predominantly expressed in basal cells [population four (P4), LinCD24+CD29hi] that have been reported to be enriched for MaSCs (3), compared with lower expression of Dll1 in luminal (P5, LinCD24+CD29lo) and stromal-enriched cells (P6, LinCD24loCD29lo) (Fig. 1, A and B). Because these data implicated a possible regulatory role of Dll1 in MaSCs, we generated keratin-14–Cre (K14-Cre)–mediated Dll1 conditional knockout (cKO) mice that target Dll1 in both basal (P4) and luminal (P5) epithelial cells (27, 28). A robust knockout of Dll1 in the mammary gland was confirmed by significant reduction of Dll1 mRNA (Fig. 1C) and Dll1 protein (Fig. 1D) in MECs of K14-Cre/Dll1f/f (Dll1cKO) mammary glands and by immunofluorescence in P4 cells (fig. S1A). Notably, a significant reduction in mammary ductal elongation and branching was observed in Dll1cKO mice compared with wild-type (WT) littermates (Fig. 1, E to G). Ki67 and EdU staining revealed fewer proliferating MECs in Dll1cKO mammary glands (Fig. 1, H and I). Staining for basal (K14) and luminal (K8) markers suggested that these cell fates were not significantly altered in the Dll1cKO MECs (Fig. 1I). The reduced ductal morphogenesis phenotype of Dll1cKO mice persisted into pregnancy, as the alveoli density in Dll1cKO mammary glands was nearly half that of WT glands at lactation day 1, leading to reduced survival of pups (fig. S1, B and C). Regardless of genotype, 80% of all pups from the Dll1cKO mothers died within 2 days of birth (fig. S1B). Histological analyses by hematoxylin and eosin (H&E) staining indicated that a smaller subset of alveoli from Dll1cKO mammary glands display features of secretory differentiation such as lipid droplets and milk production, as compared with WT alveoli (fig. S1C). We performed immunohistochemistry with Ki67 antibodies to confirm reduced proliferation in the Dll1cKO mammary glands, compared with WT mammary glands, during lactation (fig. S1C). Because the alveoli in the Dll1cKO mammary glands were lacking in lipid droplets and milk secretion, we next tested whether the proliferation defect was associated with secretory differentiation failure by staining for Np2b (Na-Pi cotransporter) protein, whose absence at parturition indicates a lack of secretory function (29). The apical membranes of secretory alveoli in Dll1cKO mammary glands showed reduced Npt2b staining compared with the WT mammary glands that exhibited intense Npt2b staining (fig. S1C). Overall, our data indicate that reduced proliferation and a block in secretory differentiation may both play a critical role in contributing to the defects in lobuloalveolar development of Dll1cKO mice.

Fig. 1 Dll1 is required for mammary gland ductal morphogenesis.

(A) FACS profile of different populations of Lin MECs, based on staining with CD24 and CD29. (B) qRT-PCR analysis of Dll1 mRNA expression in the different subpopulations of MECs shown in (A). qRT-PCR values were normalized to Gapdh. n = 5 samples. (C) qRT-PCR analysis of Dll1 mRNA expression in MECs from WT and Dll1cKO mice. n = 7 samples for WT animals and n = 9 samples for Dll1cKO animals. (D) Western blot showing Dll1 protein expression in WT and Dll1cKO MECs. (E) Representative alum carmine–stained whole-mount mammary outgrowths from WT and Dll1cKO mice at the indicated ages. (F and G) Box plot analyses of ductal elongation and branching in WT and Dll1cKO mice. Quantification of ductal branching (tertiary branch points) was measured in a defined area. n = 5 samples per genotype. (H) (Left) Ki67 staining of WT and Dll1cKO mammary gland outgrowth sections. (Right) Quantification of Ki67+ cell percentage among total epithelial cells in the field of view. n = 7 samples for WT animals and n = 5 samples for Dll1cKO animals. (I) (Left) Keratin-14 (K14), keratin-8 (K8), and EdU staining of mammary gland sections of WT and Dll1cKO mice at 5 to 6 weeks of age. Arrows indicate EdU+ cells. (Right) Quantification of EdU+ cell percentage among total epithelial cells in the field of view. n = 4 samples for each genotype. Scale bars, 2 mm in (E); 40 μm in (H) and (I). qRT-PCR data are presented as mean ± SD. *P < 0.001 by Student’s t test in (B). The P value of the box plot in (C) was computed by Mann-Whitney U test. P values in (H) and (I) were computed by unpaired and paired Student’s t tests, respectively.

Dll1 is critical for maintaining MaSC numbers

Because Dll1 is predominantly expressed in basal cell populations in which MaSCs are thought to reside (3, 5), we next probed for possible alteration of MaSC number or function in Dll1cKO mice. Fluorescence-activated cell sorting (FACS) analysis demonstrated a significant decrease in the MaSC-enriched P4 population in Dll1cKO mice (Fig. 2, A and B, and fig. S2A). Limiting dilution cleared–fat-pad repopulation assay with either total live cells (propidium iodide–negative) or lineage-negative (Lin) live cells (CD31, Ter119, and CD45) revealed a significantly reduced repopulating frequency by cells obtained from Dll1cKO mice (Fig. 2, C and D). Conversely, overexpression of Dll1 in MECs by lentiviral transduction before transplantation increased MaSC repopulation frequency (Fig. 2E and fig. S2B). Similar repopulation assays using isolated P4 and P5 cells from WT and Dll1cKO mice revealed that luminal cells (P5) from either WT or Dll1cKO mice were unable to generate ductal growth, as expected (fig. S2, C and D). Surprisingly, no significant difference was observed between WT and Dll1cKO P4 cells (Fig. 2F). Moreover, only a modest difference was observed in the serial transplant take rate of WT and Dll1cKO basal cells (fig. S2, E and F). These results indicate that the primary reason for the reduced ductal growth in the Dll1cKO mice is a reduction in the number of MaSCs rather than a defect in their function. This reduction of the MaSC-enriched P4 population was also observed during lactation (fig. S2, G and H). Taken together, our studies suggest that Dll1 plays a critical role in maintaining MaSC number during different stages of mammary gland development.

Fig. 2 Dll1 is required for maintaining MaSC activity.

(A) Representative FACS profile of Lin MECs from WT and Dll1cKO mice at 5 to 6 weeks of age. Numbers within the plots are percentages. (B) Box plot showing percentage of P4 (basal) cells in WT and Dll1cKO mice after FACS. n = 18 samples for both WT and Dll1cKO animals. See fig. S2A for individual values for the indicated groups. The P value was computed by paired t test. (C) Reconstitution efficiency of total live cells from WT and Dll1cKO mammary glands injected into cleared mammary fat pads of recipient mice. (D) Reconstitution efficiency of total Lin cells from WT and Dll1cKO mammary glands injected into cleared mammary fat pads of recipient mice. Representative alum carmine–stained mammary outgrowths from transplantation with 10,000 Lin cells are shown at bottom. (E) Reconstitution efficiency at limiting dilution of total Lin cells from WT and Dll1-overexpressing (Dll1 OE) mammary glands injected into cleared mammary fat pads of recipient mice. Representative alum carmine–stained mammary outgrowths from transplantation with 10,000 Lin cells are shown at bottom. (F) Reconstitution efficiency at limiting dilution of LinCD24+CD29hi (P4) cells from WT and Dll1cKO mouse mammary glands injected into cleared mammary fat pads of recipient mice. Representative alum carmine–stained mammary outgrowths from transplantation are shown at bottom. n indicates the number of mammary fat pad injections, as shown in (C) to (F). P values were obtained by Pearson’s chi-square test by using ELDA software. Scale bars, 2 mm in (D) to (F).

Dll1+ cells are enriched for MaSCs

To further characterize the function and expression of Dll1 in mammary glands, we used a Dll1-mCherry transgenic mouse model in which the mCherry reporter gene is driven by the Dll1 genomic regulatory sequences. In the mammary gland, the Dll1-mCherry reporter is expressed predominantly in basal cells at all developmental stages (Fig. 3A and fig. S3, C to F). FACS analysis indicated that ~12% of Lin cells are Dll1mCherry positive in virgin mice (Fig. 3B and fig. S3D), and this population increases substantially during pregnancy and lactation (fig. S3D). Notably, Dll1mCherry expression is predominantly enriched in the upper right portion of the P4 population (Fig. 3B and fig. S3, E and F).

Fig. 3 Dll1+ population is enriched in cells with MaSC activity.

(A) Immunofluorescence image of Dll1-mCherry reporter mouse mammary gland section stained with mCherry antibody. (B) (Left) Representative FACS profile of MECs from Dll1-mCherry reporter mice at 6 weeks of age. (Middle) mCherry+ cells in the Lin population. (Right) Distribution of Dll1-mCherry+ cells in different epithelial populations (left), and Dll1-mCherry+ and Dll1-mCherry cells in the P4 population (right). (C and D) Reconstitution efficiency at limiting dilution of different groups of P4 cells from Dll1-mCherry reporter mouse mammary glands injected into cleared mammary fat pads of recipient mice. For sorting of P4-Dll1hi and P4-Dll1lo, the top and bottom 10 to 12% of the population were chosen from the P4-Dll1+ cell population. n indicates the number of mammary fat pad injections. P values were obtained by Pearson’s chi-square test by using ELDA software. (E) Representative alum carmine–stained mammary outgrowths from transplantation, as indicated in (C) and (D). (F) GSEA demonstrating enriched MaSC signatures in P4-Dll1hi populations compared with P4-Dll1lo populations (26, 30). In contrast, luminal progenitor cell signatures are enriched in Dll1lo populations. NES, normalized enrichment score. Scale bars, 40 μm in (A); 2 mm in (E).

Assessment of the reconstitution potential of Dll1+ and Dll1 cells from both lineage-negative and basal cells (P4) by transplantation assay revealed that LinDll1+ cells generated mammary outgrowths more efficiently than did either LinDll1 cells or total Lin populations (fig. S4, A and B). Similarly, P4-Dll1+ cells had a much higher repopulation frequency (Fig. 3, C to E), which suggests that the Dll1+ cells represent a subset of MaSC-enriched population. Furthermore, P4-Dll1hi basal cells have increased reconstitution potential relative to P4-Dll1lo basal cells (Fig. 3, D and E). Gene set enrichment analysis (GSEA) confirmed that P4-Dll1hi cells were enriched for MaSC signatures (26, 30), whereas P4-Dll1lo cells were enriched for luminal signatures (Fig. 3F). Finally, in serial transplantation assays, both Dll1+ and Dll1hi cells continued to be more efficient in reconstitution compared with Dll1 and Dll1lo cells, respectively (fig. S4, C to E), further supporting the notion that Dll1 is enriched in the MaSC population.

Dll1+-enriched MaSCs can produce both basal and luminal cells

To examine the function of Dll1+ cells during mammary gland development, we performed a lineage tracing experiment using the previously described Dll1-GFP-IRES-Cre-ERT2 (GFP, green fluorescent protein; IRES, internal ribosomal entry site) knock-in mouse model (31). Similar to our observation in Dll1mCherry mice, Dll1GFP was predominantly expressed in basal cells, which are positive for K14 and ΔNp63 and negative for K8 (fig. S5, A to D). To trace the fate of Dll1GFP+ cells, Dll1-GFP-IRES-Cre-ERT2 mice were mated with tdTomato reporter mice (Fig. 4, A and B), and tdTomato expression was traced at different time points after the initial induction with tamoxifen in 4-week-old-mice (Fig. 4B). As expected, FACS analysis at early an time point (2 days after induction) revealed tdTomato expression predominantly in the basal compartment (Fig. 4C). Three-dimensional (3D) whole-mount staining and confocal imaging further confirmed tdTomato expression in K14+K8 basal cells (Fig. 4D). At 2 and 6 weeks after induction, tdTomato expression was observed in both basal and luminal cells, indicating that Dll1+ cells can produce both lineages (Fig. 4, E and F, and fig. S6A). Lineage tracing also confirmed that Dll1+ cells generated both basal and luminal tdTomato+ cells at pregnancy day 14.5 (Fig. 4, G and H) and in adult mammary glands (fig. S6, B to F).

Fig. 4 Lineage tracing at puberty shows that Dll1+ cells can produce both basal and luminal cell populations in mammary glands.

(A and B) Strategy for tamoxifen (TAM)–inducible Cre-mediated cell tracking using Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mice. The red box indicates Dll1-Cre–activated Tomato+ cells, which were used for lineage tracing of Dll1+ cells. (C) FACS plot of MECs from Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mouse mammary glands after 2 days of induction with TAM, showing the percentage of tdTomato+ cells in various mammary epithelial populations, based on staining with CD24 and CD29. (D) (Left and middle) Whole-mount 3D staining images showing Tomato+ cells in basal cells. (Right) Staining with K14, K8, and Tomato antibodies on confocal sections on Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mouse mammary glands after 2 days of TAM treatment. Arrows indicate Tomato+K14+ basal cells. DAPI, 4′,6-diamidino-2-phenylindole. (E) FACS plot of MECs from Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mouse mammary glands after 2 weeks of induction with TAM, showing the percentage of tdTomato+ cells in various mammary epithelial populations, based on staining with CD24 and CD29. (F) (Left) Whole-mount 3D staining image showing Tomato+ cells in both luminal and basal cells, suggesting that Dll1+ cells can generate both basal and luminal cells. (Middle and right) Staining with K14, K8, and Tomato antibodies on confocal sections of Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mouse mammary glands after 2 weeks of TAM treatment. White arrows indicate Tomato+K14+ basal cells; red arrows indicate Tomato+ K8+ luminal cells. (G) FACS plot of MECs from Dll1-GFP-IRES-Cre-ERT2; ROSA-tdTomato mouse mammary glands during pregnancy after induction with TAM, showing the percentage of tdTomato+ cells in various mammary epithelial populations, based on staining with CD24 and CD29. (H) (Left) Whole-mount 3D staining image showing Tomato+ cells in both luminal and basal cells, suggesting that Dll1+ cells can produce both basal and luminal cells. (Middle and right) Staining with K14, K8, and Tomato antibodies on confocal sections of Dll1-GFP-IRES-Cre-ERT2;ROSA-tdTomato mouse mammary glands at day 14.5 of pregnancy. White arrows indicate Tomato+K14+ basal cells; red arrows indicate Tomato+ K8+ luminal cells. n = 5 samples per developmental stage. Scale bars, 40 μm in (D), (F), and (H).

Mammary gland macrophages have distinctive molecular properties and are regulated by Dll1+ MaSCs

Because Notch signaling is involved in intercellular signaling, we next determined whether Dll1 knockout in MECs affected specific stromal cell populations in the mammary glands. FACS analysis showed reduced F4/80+ macrophage and moderately reduced PDGFRα+ fibroblast populations in Dll1cKO mammary glands compared with WT glands (Fig. 5, A to C), whereas no significant difference was observed for CD31+ endothelial cells (Fig. 5, B and C). Immunostaining further confirmed reduced F4/80+ macrophages in Dll1cKO terminal end buds (TEBs) and ducts at different developmental time points (Fig. 5D and fig. S7, A to I), with corresponding reduction of P4 cells (fig. S7, A to I) and increased apoptotic activity in macrophages (Fig. 5E). Immunofluorescence analysis of mammary gland sections and mammospheres from a 3D in vitro coculture system demonstrated that Dll1+ basal cells localized close to F4/80+ stromal cells (Fig. 5, F, G, and H), suggesting potential cross-talk between the two populations via juxtacrine or paracrine signaling. Using the 3D coculture assay, we next tested the impact of macrophages on Dll1+ MaSC activity. Notably, mammary gland macrophages can induce MaSC activity, as reflected by increased mammosphere numbers, whereas peritoneal macrophages could not (Fig. 5I), indicating a tissue-specific function. Furthermore, enhancement of stem cell activity was much more prominent in P4-Dll1+ cells than in P4-Dll1 cells (Fig. 5I), suggesting that MaSCs depends on Dll1 to engage and respond to mammary gland macrophages.

Fig. 5 Mammary gland macrophages have distinctive molecular properties and are regulated by Dll1+ MaSCs.

(A) FACS plot of MECs from WT and Dll1cKO mammary glands, based on staining with F4/80 (macrophages) and PDGFRα (fibroblasts). (B) Histogram from FACS analyses showing CD31+ endothelial cells in WT and Dll1cKO mammary glands. (C) Box plots showing quantification (percentage) of F4/80+ macrophage (Mϕ) , PDGFRα+ and CD31+ stromal cell populations in WT and Dll1cKO mammary glands, based on staining with respective antibodies. Mann-Whitney U test was used for all of these analyses. n = 5 samples per genotype. (D) F4/80 antibody staining in WT and Dll1cKO mammary gland sections shows fewer F4/80+ cells at TEBs and ducts of Dll1cKO mice relative to WT animals. n = 3 samples per genotype. (E) Box plots showing quantification (percentage) of CD45+ F4/80+ macrophages that are positive for cleaved caspase-3 activity in WT and Dll1cKO mammary glands, based on staining with respective antibodies. n = 5 samples for WT mice and n = 6 samples for Dll1cKO mice. (F) Immunofluorescence image of Dll1-mCherry reporter mouse mammary gland section at 6 weeks shows juxtaposition of Dll1-mCherry+ cells (green) with F4/80+ (red) macrophages. Dll1mCherry+ cells were indirectly detected by using a secondary antibody for mCherry that was conjugated to Alexa 488 green fluorescent dye. Macrophages were stained with F4/80 antibody, which conjugated to Alexa 568 red fluorescent dye. (G) Coculture mammosphere assay of P4 cells from WT mice (bright field) with macrophages from Actin-dsRED mice (red). (H) Confocal images of mammospheres of P4 cells from Actin-dsRED mice with macrophages from Actin-GFP mice (green) mammary glands, showing juxtaposition of basal cells (red) with macrophages (green) in mammospheres. (I) Number of mammospheres formed by P4-Dll1+ and P4-Dll1 cells with and without macrophages from the mammary gland or the peritoneum, respectively. n = 3 samples. Error bars indicate SD. Student’s t test was used for statistical analysis. **P < 0.01. (J and K) GSEA showing enrichment of Notch and Wnt signaling pathway signatures in mammary gland macrophages compared with peritoneal macrophage populations. (L) Fold change (FC) in gene expression of the most differentially expressed Notch and Wnt genes between mammary gland (M) and peritoneal (P) macrophage populations from WT mice. (M to O) Western blots showing Notch4, Notch3, and Notch1 protein expression in the sorted population of mammary resident macrophages (M-Mϕ), resident peritoneal macrophages (P-Mϕ) and activated peritoneal macrophages (PAct-Mϕ), respectively, from 6-week-old virgin mice. Scale bars, 40 μm in (D); 20 μm in (F) to (H). The same β-actin loading control was used in (M) to (O).

A gene expression study showed that the overall gene signature of the mammary macrophages is more similar to that of the resting peritoneal macrophages than to that of activated peritoneal macrophages (fig. S8). GSEA showed that, compared with peritoneal macrophages, mammary gland macrophages are enriched for Wnt- and Notch-related gene signatures (Fig. 5, J and K), including several Wnt ligands and Notch receptors (Fig. 5L). Elevated expression of Notch1, -3, and -4 in mammary macrophages compared with peritoneal macrophages was further confirmed by Western blot analysis (Fig. 5, M to O). Together, these data suggest that MaSCs depend on Dll1 to engage and respond specifically to resident macrophages in the mammary gland.

Depletion of macrophages reduces function of Dll1+ MaSCs

To investigate Dll1- and Notch-dependent function of macrophages within the MaSC niche, we first used clodronate liposomes (CLs) to deplete macrophages in Dll1-mCherry transgenic mice (32). Systemic ablation of macrophages decreased Dll1mcherry+ MaSC numbers (fig. S9, A and B) and increased their apoptosis (fig. S9, C and D). Next, we performed a cotransplantation assay by injecting Dll1mcherry+ MaSCs into cleared mammary fat pads with either clodronate-containing or control liposomes. We observed a nearly complete inhibition of reconstitution in the tissue injected with CL-containing Dll1mcherry+ MaSCs compared with control tissue, indicating the dependence of MaSCs on macrophages (fig. S9, E and F).

To more specifically test whether macrophages are necessary for Dll1+ MaSC activity, we used two additional approaches to deplete macrophages in vivo: (i) Csf1r-blocking antibody treatment (33) (Fig. 6, A to D), and (ii) macrophage Fas-induced apoptosis (MaFIA) mice (34) (Fig. 6, E to H), in which administration of the drug AP20187 induces apoptosis and depletes macrophages (34). Both Csf1r-blocking antibody treatment in WT mice and AP20187 treatment in MaFIA mice significantly reduced the number of macrophages (Fig. 6, C, D, G, and H) without affecting dendritic cells and neutrophils (fig. S9G) and blocked the repopulation of the mammary gland by Dll1+ P4 cells (Fig. 6, A, B, E, and F).

Fig. 6 Depletion of macrophages or genetic knockout of Notch signaling in macrophages reduces stem cell activity of Dll1+ basal cells.

(A) Take rate of transplantation with 200 P4-Dll1+ and P4-Dll1 cells from Dll1-mCherry mouse mammary glands. Recipient mice were treated with immunoglobulin G (IgG) (control) and Csf1r antibody (Csf1r Ab) (500 μg per mouse) for 4 to 5 weeks. (B) Representative alum carmine–stained whole-mount mammary outgrowths from populations indicated in (A). (C and D) Quantification of macrophages from mammary glands (MG) and peripheral blood from control and Csf1r antibody–treated mice. FACS was performed by using CD45 and F4/80 antibodies to detect macrophages (n = 5 mice per condition). (E) Take rate of transplantation with 500 P4-Dll1+ and P4-Dll1 cells from Dll1-mCherry mouse mammary glands into MaFIA recipient mice. Recipient MaFIA mice were treated with either vehicle or AP20187 (5 mg per kilogram of body weight) for 3 weeks, and mammary outgrowths were harvested at 5 weeks. (F) Representative alum carmine–stained whole-mount mammary outgrowths from populations indicated in (E). (G and H) Quantification of macrophages from mammary glands and peripheral blood from control and MaFIA-treated mice. FACS was performed by using CD45 and F4/80 antibodies to detect macrophages (n = 5 mice per condition). (I) Representative alum carmine–stained whole-mount mammary outgrowths from WT and RbpjkcKO (CD11c-Cre;Rbpjkf/f) mice (35) at 5 to 6 weeks. (J to L) Box plot analyses of ductal elongation and branching and terminal end bud (TEB) counts in WT and RbpjkcKO mice. Quantification of ductal branching (tertiary branch points) was measured in a defined area. (M) Box plot showing percentage of P4 (basal) cells in WT and RbpjkcKO mice. In (J) to (M), n = 5 samples for both WT and RbpjkcKO animals. (N) Take rate of transplantation using a mixed population of 500 P4-Dll1+ and 2000 mammary macrophages. Sorted mammary macrophages from Actin-GFP mice were infected with either control lentivirus or Rbpjk shRNAs (KD1 and KD2). P4-Dll1+ cells were obtained from sorting of Dll1-mCherry mouse mammary glands. P = 0.0266; Fisher’s exact test. See schematic in fig. S9H for additional details of the experimental design. (O) Representative carmine alum–stained images of transplants of different groups from populations indicated in (N). Mann-Whitney U test was used to obtain P values in (C), (D), (G), (H), and (J) to (M). Scale bars, 1 mm in (B), (F), and (O); 2 mm in (I).

We further used two in vivo models to investigate the importance of Notch signaling in macrophages for sustaining MaSC activity. First, we used the previously reported RbpjkcKO (CD11c-Cre; Rbpjk floxed) mouse model (35) in which Rbpjk, a mediator of Notch signaling, is conditionally deleted in macrophages. Five- to six-week-old RbpjkcKO mice showed a significant reduction in mammary ductal elongation, branching, and TEB counts compared with their WT littermates (Fig. 6, I to L). This phenotype was also associated with a decreased basal (P4) population (Fig. 6M), phenocopying Dll1cKO mice. Next, we used an ex vivo transplant method (fig. S9H) in which mammary gland macrophages were isolated from actin-GFP mice, and Rbpjk was knocked down by lentiviral transduction (fig. S9I) by using two previously reported short hairpin RNAs (shRNAs) (36). Rbpjk-KD and control mammary macrophages were then mixed with Dll1mcherry+ P4 cells and transplanted into recipient NSG mice, which have defective macrophages. The take rate of mammary outgrowths was significantly reduced when P4-Dll1mcherry+ cells were mixed with Rbpjk KD macrophages compared with control macrophages (Fig. 6, N and O, and fig. S9, H and I). Taken together, these studies demonstrate a functional dependence of Dll1+ MaSCs on mammary macrophages through Notch signaling.

Dll1 regulates Notch signaling in neighboring macrophages

We developed an in vitro coculture assay (fig. S10, A and B) to further investigate the molecular role of Dll1-mediated Notch signaling between macrophages and Dll1+ MaSCs. The addition of P4-Dll1mcherry+ cells to the culture induced Hes1 and Hey1 expression in F4/80+ macrophages but not in fibroblasts and endothelial cells (fig. S10, C and D). Hes1 and Hey1 expression was more pronounced in Dll1mCherry+ basal cells than in Dll1mCherry− basal cells when cocultured with macrophages (fig. S10, E and F). Such Dll1-dependent Notch downstream gene activation was suppressed with a Dll1-blocking monoclonal antibody (Fig. 7A). In F4/80+ macrophages from WT mammary glands, Notch2 and Notch3 are the most abundantly expressed Notch receptors (fig. S10F). Treatment of the coculture of P4-Dll1+ mammary stem cells and macrophages with either Notch2- or Notch3-blocking antibody reduced Hey1 expression (Fig. 7B), indicating that Notch2 and Notch3 mediate Dll1-dependent cross-talk between MaSCs and macrophages.

Fig. 7 Dll1-mediated cross-talk between MaSCs and macrophages promotes Wnt ligand expression in macrophages to support MaSC activity.

(A and B) Hey1 mRNA levels in F4/80+ cells after coculture with P4-Dll1+ cells with and without blocking antibody against Dll1, Notch2, and Notch3 receptors. (C) Mammosphere assay with P4 cells from WT and Dll1cKO MECs with and without WT and Dll1cKO macrophages. n = 5 samples. (D) Mammosphere assay with P4-Dll1+ cells with and without macrophages and treatment of antibodies against Dll1, Notch2, and Notch3. n = 3 samples. (E) Fold change in gene expression of the most differentially expressed genes encoding secreted factors or extracellular proteins between macrophage populations from WT and Dll1cKO mammary glands. (F to H) Wnt3, Wnt10a, and Wnt16 mRNA levels in F4/80+ cells after coculture with P4-Dll1+ cells with and without blocking antibody against Dll1, Notch2, and Notch3. n = 3 samples. (I to K) Representative immunofluorescence images of coculture cells (macrophages cultured for 3 days followed by addition of P4-Dll1+ cells for 5 h) stained with Wnt3, Wnt10a, and Wnt16 antibodies. The control was macrophage cultured alone without P4-Dll1+ cells. (L) Quantification of Wnt3, Wnt10a, and Wnt16 immunofluorescence intensity in indicated groups from (I) to (K). (M) Mammosphere assay of WT P4 cells with and without coculture with macrophages along with Wnt inhibitor Dkk1. n = 4 samples. For macrophage isolation, a combination of F4/80 and CD140 antibodies was used. (N) Model showing cross-talk of a Dll1+ MaSC–enriched population with macrophages through Notch and Wnt signaling. All qRT-PCR experiments were performed three times. Data are presented as mean ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05 by Student’s t test in (A) to (D), (F) to (H), (L), and (M). Scale bars, 10 μm in (I) to (K).

To examine the functional importance of Dll1-Notch signaling within the MaSC-macrophage niche, we again used mammosphere coculture assays. When macrophages from WT mice were mixed with either WT or Dll1cKO P4 cells, there was a significant increase in mammosphere number (Fig. 7C), suggesting a MaSC-promoting property of mammary gland macrophages. Conversely, macrophages from Dll1cKO mice (MϕcKO) were less competent in promoting mammosphere formation of either WT or Dll1cKO P4 cells, indicating an altered cellular property of the macrophages from Dll1cKO mammary glands. Consistent with the role of Dll1 and Notch2 and -3 in mediating the cross-talk between MaSCs and macrophages, treatment of the mammosphere coculture by antibodies against Dll1, Notch2, or Notch3 reduced the number of mammospheres (Fig. 7D). Overall, our data indicate a Dll1-mediated Notch signaling pathway between MaSCs and macrophages that is crucial for supporting MaSC activity.

Dll1-dependent expression of Wnt ligands in macrophages

To gain mechanistic insight as to how macrophages dictate the cell fate of MaSCs, we performed global transcriptomic analysis of F4/80+ macrophages isolated from WT and Dll1cKO mammary glands. Focusing on extracellular secreted factors and cytokines, we found that among the 10 most differentially expressed genes were three genes coding for Wnt ligands: Wnt10A, Wnt16, and Wnt3 (Fig. 7E). These data are consistent with our earlier finding (Fig. 5, K and L) showing that mammary macrophages are enriched for Wnt signaling genes as compared with peritoneal macrophages. Quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) and immunofluorescence analyses of Wnt3, Wnt10a, and Wnt16 in the coculture system further confirmed Dll1-Notch2– or Dll1-Notch3–dependent stimulation of Wnt ligand expression in macrophages (Fig. 7, F to L). Furthermore, stimulation of mammosphere forming activity of P4 cells by macrophages was largely blocked by the presence of the Wnt signaling inhibitor Dkk1 (Dickkopf-1) in the coculture (Fig. 7M). These results indicate that the regulation of the MaSC population by macrophages is likely mediated by increased Wnt ligand production by macrophages in response to Dll1-Notch signaling.

Discussion

Intercellular signaling between stem cells and stromal cells within the stem cell niche dictates stem cell number and function, including self-renewal activity. Although other stem cell niches have been extensively studied, the current inability to identify MaSCs within their associated stromal niche has hindered similar studies in the mammary gland. In our study, we first showed that Dll1 is a marker and crucial regulator of MaSCs. Using transplantation assays, we showed that Dll1+/hi cells are enriched for MaSCs with increased regenerative potential. Further, we used lineage tracing experiments to confirm that Dll1+ basal cells can generate both basal and luminal cells. However, similar to other published lineage tracing studies (20, 37), we cannot completely rule out the possible contribution of a small fraction of Dll1+ luminal cells to the luminal population expansion.

Macrophages have been reported to be components of the spermatogonial and hematopoetic stem cell niches (38, 39). They have also been shown to play a role in mammary gland development (13); however, the exact signaling mechanism between macrophages and MaSCs is not known. In our study, we found that the F4/80+ macrophage population was reduced in Dll1cKO mammary glands in different developmental stages, probably owing to increased cell death as seen by cleaved caspase-3 activity. It is also possible that a lower expression level of Csf1, a critical cytokine for macrophage differentiation, in mammary gland macrophages of Dll1cKO mice compared with WT mice (Fig. 7E) might also contribute to the lower number of mature macrophages. Notably, the reduced ductal elongation and branching phenotype of Dll1cKO mammary glands is comparable to the mammary gland defects observed in the Csf1 knockout mice with macrophage deficiency (12). Similarly, we also observed that Dll1+ cells could not regenerate mammary glands when macrophages were depleted by CL or Csf1r antibody treatment or by AP20187 injection in the MaFIA mice. Moreover, by using CD11c-Cre;RbpjkcKO mice and lentiviral-mediated Rbjpjk knockdown in macrophages, we further showed the dependence of Dll1+ MaSCs on Notch signaling in mammary macrophages. These studies thus delineate macrophages as one of the important components of the mammary stem cell stromal niche. Gene expression analysis identified several Wnt and Notch signaling genes enriched in mammary macrophage populations compared with peritoneal macrophages, indicating the distinctive ability of mammary gland macrophages to sustain the MaSC pool. These results collectively indicate reciprocal interactions between macrophages and MaSCs: Dll1-Notch signaling from MaSCs to macrophages maintains the number and niche-related activity of the macrophages; conversely, the macrophageal niche is crucial for sustaining the MaSC pool.

Consistent with these notions, we established and used a 3D coculture assay to show that Dll1+ MaSC-enriched basal cells interact with stromal macrophages through Notch2 and -3 receptors. This organoid coculture system aims to mimic the point at which there is substantial contact between MaSCs and macrophages. With the use of this system, coculturing MaSCs with macrophages resulted in a significant increase in stem cell activity of Dll1+ cells. Conditional knockout of Dll1 in the MECs not only renders the Dll1cKO basal cells less responsive to macrophage activation but also reduces the potency of macrophages from Dll1cKO mice in supporting MaSC function. Macrophages isolated from Dll1cKO mice have reduced expression of several Wnt family ligands, including Wnt3, Wnt10A, Wnt16, which suggests that Dll1-dependent Notch signaling is responsible for promoting the expression of the Wnt ligands in the macrophages that are in close contact with MaSCs.

Although there is strong evidence that Wnt signaling is important for MaSCs, the source of the Wnt ligands was previously unknown. Our study shows that mammary gland macrophages produce Wnt ligands after Notch signaling is activated by Dll1 from MaSCs, which in return induces the MaSC activity. This situation is somewhat reminiscent of the crypt stem cell niche, where Paneth cells produce large amounts of Wnt3 to maintain stem cells and where stem and Paneth cells communicate through Notch-delta signaling (40, 41). Stroma mediated Wnt–β-catenin signaling has also been reported to promote the self-renewal of hematopoietic stem cells (42). Notably, we have previously reported a high level of ΔNp63 expression in MaSCs, which transcriptionally activates the expression of Wnt receptor Fzd7 (26). Therefore, not only are ΔNp63 and Dll1 markers of MaSCs, but they also functionally support MaSC activity through sustaining a locally enriched Wnt signaling environment.

Our study establishes macrophages as important cellular components of the MaSC niche through intercellular coupling of Notch and Wnt signaling (Fig. 7N). It is possible that additional niche stromal cells may also play a central role in MaSC regulation, which requires future exploration. In the context of Dll1-mediated Notch signaling in MaSC-macrophage cross-talk, we found that Dll1 produced from MaSCs activates Notch signaling in macrophages to enhance the expression of Wnt ligands, which in turn supports Wnt signaling in MaSCs to maintain stem cell activity (Fig. 7N). Because Dll1-Notch signaling requires direct cell-cell contact and Wnt ligands mostly act as short-range intercellular signals, the Dll1-mediated coupling of Notch-Wnt signaling ensures a spatially delimiting mechanism for localized MaSC–macrophageal niche interaction (23). As Notch and Wnt pathways have been reported to be key oncogenic pathways in breast cancer and macrophages are a major component of the tumor microenvironment, future studies of Notch-Wnt–dependent interaction between MaSCs and macrophages may provide insights into tumor initiation and progression in breast cancer.

Materials and methods

Animal studies

Animal procedures were conducted in compliance with Institutional Animal Care and Use Committee (IACUC) of Princeton University, University of Pennsylvania, and Memorial Sloan Kettering Cancer Center. Dll1 floxed mice (28), Dll1-GFP-IRES-Cre-ERT2 mice (31), and CD11c-Cre;RbpjkcKO mice (35) have been described previously. The Dll1-mCherry transgenic mice were generated using a genomic BAC clone with mCherry cDNA inserted after the start codon of Dll1. tdTomato mice [B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J], Actin-GFP, Actin-dsRED mice, and MaFIA mice were obtained from Jackson Laboratory. For all animal experiments, control littermate animals were utilized. For cleared fat-pad injection experiment, C57/B6, athymic nude and NSG mice at 3 weeks old were anaesthetized and a small incision was made to reveal the mammary gland. MECs as specified in each experiment were injected into cleared inguinal (#4) mammary fat pads according to the standard procedures (43, 44).

Limiting dilution assay (LDA)

Single-cell suspension of primary MECs from WT and Dll1cKO mammary glands at 5 to 7 weeks were sorted using the lineage (CD31, Ter119, and CD45), CD24, and CD29 markers to obtain the MaSC-enriched P4 population (LinCD24+CD29hi), which was then injected into cleared mammary fat pads. The outgrowths were analyzed at 6 to 8 weeks posttransplantation. Transplantation was performed with indicated number of cells resuspended in 50% Matrigel and 50% PBS. For transplantation assay with CL treatment, assay was performed following protocol from the previously published work (13). For transplantation assay using Csf1r-blocking antibody, mice were pretreated once with either control IgG or blocking antibody followed by treatment every 3 days with antibodies at a concentration of 500 μg per mouse. 200 P4-Dll1+ or P4-Dll1 basal cells were used for transplantation. Treatment continued for 4 weeks and mice were harvested at 6 weeks after injection. For transplantation using MaFIA mice as recipients, 500 P4-Dll1+ or 500 P4-Dll1 basal cells were injected into cleared mammary gland of MaFIA mice and these mice were treated (by IP injection) with AP20187 at 5 mg/g (Ariad Pharmaceuticals) every 3 days, which leads to depletion of macrophages. Similar to Csf1r antibody experiment, mice were pretreated once with the control or drug AP20187 at the concentration of 10 mg/kg. Treatment continued for 3 weeks and mice were euthanized to examine reconstitution of mammary gland at 5 weeks after injection. For the ex vivo transplant assay with mixture of mammary macrophages with P4-Dll1mCherry+ cells, see schematic in fig. S9H for the detailed process. Frequency of MaSCs in the transplanted cell suspension was calculated using L-calc software (StemCell Technologies) or ELDA (extreme limiting dilution assay) (30, 45). Single-hit model was also tested using ELDA and value of slope was 1. MaSC abundances were assumed to follow a Poisson distribution in LDAs, and generalized linear models utilizing a log-log link function were used to derive repopulation frequency parameters. Self-renewal activity of MaSCs after transplantation was tested by their ability to regenerate functional mammary glands in virgin mouse.

Clondronate liposome (CL) assay

The CLs are nontoxic until ingested by macrophages. Once ingested, they are then broken down by liposomal phospholipases to release the drug that subsequently induces cell death in macrophages by apoptosis (32). For systemic treatment of Dll1mCherry+ reporter mice, the animals were treated with CL (150 to 170 μl) at 5 weeks of age (mouse body weight ~15 to 17 g) every other day for 1 week before the mammary glands were harvested. For mammary fat-pad reconstitution assay, P4-Dll1mCherry+ cells were mixed with or without CL following the published procedure (13) and then injected into cleared mammary fat pad of C57/B6 mice. Transplants were harvested 6 weeks postinjection.

Mammosphere assays

Stem cells from the mammary gland have been successfully maintained and passaged in vitro as spheroids in suspension. Cells were cultured as previously described (36). For coculture mammosphere assay with P4 basal cells (WT or Actin-dsRED) and macrophages (WT or Actin-dsRED or Actin-GFP), 5000 P4 cells were mixed with 20,000 macrophages and grown in low adherent plate in mammosphere media (36). This 1:4 ratio of P4:macrophage coculture was determined to be the optimal in vitro coculture condition in which macrophages strongly enhance the mammosphere forming activity of P4 cells.

Cloning, viral production, and infection

The pLEX plasmid (Open Biosystems) expressing Dll1 cDNAs was generated by routine molecular cloning techniques. All plasmids were packaged into virus using HEK293-T cells as packaging cell lines and helper plasmids VSVG and dR8.9 following standard protocols. Primary cells were spin-infected with virus-containing media supplemented with 2 μg/ml polybrene for 2 hours at 1000 g at 4°C and then transplanted. Rbpjk shRNAs (purchased from Open Biosystems Inc.) were previously validated in our earlier studies (36). Macrophages from Actin-GFP mice were sorted using cocktail of F4/80 and CD140 antibodies and were spin infected similar to MECs using lentivirus.

Coculture assay

Briefly, various stromal cell populations from WT or Actin-GFP+ mice mammary glands were isolated by sorting and plated on gelatin-coated plates for 3 to 5 days. Dll1 (0.75 μg/ml) or Notch2 or Notch3 (1.5 μg/ml) blocking antibodies were added alone or in combination followed by the addition of control (no P4 cell), P4- Dll1mCherry+ cells (P4-Dll1+) or P4- Dll1mcherry− cells (P4-Dll1) for 90 min. Cells are then washed, trypsinized and sorted for either mCherry+ and mCherry- population or mCherry+ and GFP+ population followed by RNA isolation for gene expression analysis. For IF with Wnt antibodies, macrophages were cocultured for 5 hours with P4-Dll1mCherry+ cells. Coculture was washed extensively to remove P4 cells. Attached macrophages were stained with respective Wnt antibodies.

Protein extraction and Western blot analysis

Proteins were extracted from primary epithelial cell cultures and cell lines in RIPA buffer as previously described (27). Western blot analysis was performed using the standard protocol. Antibodies and dilutions used are listed in table S1.

Histological analysis, immunohistochemistry (IHC), and immunofluorescence (IF)

For histological analysis, mammary gland specimens were processed as previously described (36). Antibodies and dilutions used are listed in table S1. DAPI was used to stain nuclei. Confocal images were taken using a Nikon A1 confocal microscope or Nikon TiE microscope. For immunofluorescence analysis of sorted P4 cells, cells were attached to slides by gentle cytospin followed by immunofluorescence which was performed after fixing and permeabilizing the cells for 20 min at RT. Dll1 antibody is listed in supplementary table S1.

Flow cytometry and FACS sorting

Single MECs were obtained from mammary glands following the published protocol (3, 4, 26, 36). Briefly, MECs were stained with a combination of lineage, CD24, and CD29 antibodies (3) for 20 to 30 min on ice following the published protocol. FACS analysis was performed using the LSRII Flow Cytometer (BD Biosciences) and data were analyzed using FlowJo software (TreeStar, Inc). For sorting cells, FACS Vantage or Aria II instruments were used. For cleaved caspase-3 assay, MECs were fixed and then stained with antibodies following manufacturer’s protocol (BD Biosciences). For isolation and or FACS analysis of macrophages from different tissues, either CD140 and F4/80 antibody cocktail or CD45 and F4/80 antibody cocktail was used. For DCs and neutrophils, cocktail of CD45, CD11b, Gr1, and CD11c antibodies were used. Live cells were gated out using either DAPI or PI. Details about all FACS related antibodies are listed in table S2.

EdU assay

Mice were intraperitoneally injected with EdU (0.2 mg per 10 g body weight, Invitrogen) 2 or 12 h before mammary gland harvest. EdU was visualized using Click-it Imaging reagents (647 and 488) from Invitrogen following the protocol from manufacturer. For EdU assay in FACS along with other antibodies, samples were first stained with CD24, CD29, Ter119, CD45, and CD31 antibodies, fixed and then stained with EdU following the protocol from manufacturer (Invitrogen). For immunofluorescence, paraffin embedded sections were first rehydrated using standard protocol and then stained with EdU followed by other antibodies following the manufacturer’s instructions.

qRT-PCR analyses

Total RNA was isolated from primary cells using Qiagen RNA extraction kit in accordance with the manufacturer’s instructions. Real-time RT-PCR was performed on ABI 7900 96 HT series and StepOne Plus PCR machines (Applied Biosystem) using SYBR Green Supermix (Bio-Rad Laboratories). The gene-specific primer sets were used at a final concentration of 0.2 μM, and their sequences are listed in table S3. All qRT-PCR assays were performed in duplicate in at least three independent experiments using three different tissue samples.

Microarray analysis

The P4, P5, and P6 subpopulations of MECs were isolated from the mammary glands (four mammary glands from each group) of virgin mice. MECs were isolated using FACS as described in (46). The sorted P4 cells from Dll1-mCherry mice mammary gland or macrophages from WT and Dll1cKO mice (C57/B6 strain) at 5 to 6 weeks of age were prepared as described. For activated peritoneal macrophages, macrophages were activated with Bio-Gel P-100 and obtained from C57/B6 mice. RNA was collected from these samples using the RNAeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The gene expression profiles of various populations of macrophages from the WT and Dll1cKO mice or P4 (Dll1hi or Dll1lo) were determined using Agilent mouse GE 8x60k two-color microarrays system (Agilent, G4852A), following the manufacturer’s instructions. Briefly, the RNA samples and universal mouse reference RNA (Agilent 740100) were labeled with CTP-cy5 and CTP-cy3, respectively, using the Agilent Quick Amp Labeling Kit. Labeled testing and reference RNA samples were mixed in equal proportions, and hybridized to the mouse GE 8x60K array. The arrays were scanned with an Agilent G2505C scanner and raw data was extracted using Agilent Feature Extraction software (v11.0). Data was analyzed using the GeneSpring 13 software (Agilent). The expression value of individual probes refers to the Log2(Cy5/Cy3) ratio.

Gene set enrichment analysis (GSEA)

GSEA v2.2.0 was used to perform the GSEA on various functional and/or characteristic gene signatures (47, 48). Normalized microarray expression data were rank-ordered by differential expression between cell populations and/or genetic background as indicated, using the provided ratio of classes (i.e., fold change) metric. Two independent MaSC specific gene signatures were used to characterize MaSC characteristics. Both are defined by significantly up-regulated genes (P < 0.05 and FC > 3) in MaSC-enriched subpopulations from MECs of WT mice. Among which, the “MaSC signature and luminal cell signature from Chakrabarti et al., 2014” (Fig. 3F) is derived from the microarray data collected from our lab as described in previous study (26) (GSE47493). The genes showing >3 folds up-regulation in P4 comparing to both P5 and P6 of WT mice were included in the MaSC gene set. For luminal signature, the genes showing >3 folds up-regulation in P5 comparing to both P4 and P6 of WT mice were included in the luminal gene set. The other MaSC and luminal cell signature is derived from published dataset (30). For gene expression in macrophages, the genes showing >3 folds up-regulation in mammary resting macrophages comparing to resting peritoneal macrophage of WT mice were included.

Lineage tracing

Lineage tracing experiment was performed following protocols previously described (5). In brief, tdTomato reporter expression in Dll1-GFP-IRES-Cre-ERT2/ROSA-tdTomato mice were induced by intraperitoneal injection of 1.5 mg of tamoxifen (75 μl of 20 mg/ml) diluted in corn oil (Sigma) at the indicated age during puberty or adulthood and kept for different time points followed by whole mount or FACS analysis.

Statistical analysis

Results were generally reported as mean ± SD (standard deviation) as indicated in the figure legend. For comparisons of central tendencies, normally distributed datasets were analyzed using unpaired (with the exception of analyses of cellular populations from paired samples) two-sided Student’s t tests under assumption of equal variance. Non-normally distributed datasets were analyzed using nonparametric Mann-Whitney U tests. To adjust for host effects, paired two-sided Student’s t tests assuming equal variance were used for experiments in which cellular populations were compared following matched control and experimental cell types (Figs. 1I and 2B and figs. S2F and S9, A to C). Statistical analyses specific to LDAs and GSEA are described above. All the experiments with representative images (including Western blot, FACS plot, histology, and immunofluorescence) have been repeated at least thrice and representative images were shown. For animal studies, no statistical test was performed to predetermine the sample size. Animals were excluded only if they died or had to be euthanized because of moribund conditions following the IACUC protocol.

Accession numbers for datasets

Microarray data reported herein have been deposited at the NCBI Gene Expression GSE77504.

Supplementary Materials

References and Notes

Acknowledgments: We thank L. King (University of Pennsylvania) for critical reading of the manuscript and helpful discussions. Funding: This work was supported by a DOD Postdoctoral Fellowship (BC103740) and a NCI-K22 grant (K22CA193661) to R.C.; a Susan G. Komen Fellowship (PDF15332075) to T.C.-T.; grants from the Brewster Foundation, the Breast Cancer Research Foundation, DOD (BC123187), and NIH (R01CA141062) to Y.K; and NIH grants (R01 CA198280-01 and P30 CA008748) to M.O.L. This research was also supported by the Genomic Editing and Flow Cytometry Shared Resources of the Cancer Institute of New Jersey (P30CA072720). Author contributions: R.C. and Y.K. designed all experiments. R.C., S.K., T.C.-T., X.H., A.C., J.H., and J.P. performed the experiments. C.D. and J.J.G provided technical advice and helped with FACS analysis and sorting. Y.W. performed all microarray and statistical analyses. B.N. and M.O.L. participated in RbpjkcKO-related experiments. J.G., J.H.v.E., I.A., and H.C. provided mouse strains and advice. R.C and Y.K. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing interests: The authors declare no competing interests. Data and material availability: All data needed to understand and assess the conclusions of this research are available in the main text and supplementary materials. Microarray data reported herein have been deposited at the NCBI Gene Expression Omnibus GSE77504.
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