Tissue-resident macrophages promote extracellular matrix homeostasis in the mammary gland stroma of nulliparous mice
- Department of Laboratory Medicine and Pathology, University of Minnesota, United States
- University of Minnesota Supercomputing Institute, University of Minnesota, United States
- Comparative and Molecular Biosciences Graduate Program, University of Minnesota, United States
- Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane School of Medicine, United States
- Masonic Cancer Center, University of Minnesota, United States
- Center for Immunology, University of Minnesota, United States
Figures
Figure 1 with 2 supplements
Identification of Lyve-1+ macrophages in the mammary gland.
(A) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+ and CD45– Lyve-1+ cells by flow cytometry. (B) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+CD11b+F4/80…
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Figure 1—source data 1
Source data for graphs in panels A and B.
- https://cdn.elifesciences.org/articles/57438/elife-57438-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Identification of Lyve-1+ macrophages in the mammary gland.
(A) Gating of flow cytometry for CD45+ and CD45– Lyve-1+ cells. (B) Gating of flow cytometry for CD45+CD11b+F4/80+Lyve-1– and CD45+CD11b+F4/80+Lyve-1+ cells.
Figure 1—figure supplement 2
Localization of Lyve-1+ macrophages in the mammary gland.
Image of a mammary gland from a 6-week-old female mouse (n = 3) stained for F4/80 (red), Lyve-1 (green) and DAPI (blue). Insets show examples of the mammary gland capsule (a–d) and lymph nodes (e–h).
Figure 2 with 1 supplement
Identification of a distinct Lyve-1+ macrophage subpopulation by transcriptional profiling.
(A) Heat map of RNA-seq analysis of CD45+CD11b+F4/80+Lyve-1– and CD45+CD11b+F4/80+Lyve-1+ cells isolated from 6-week-old (lane 1) and 10-week-old (lanes 2–3) mice. Genes shown have an adjusted …
Figure 2—figure supplement 1
Identification of a distinct Lyve-1+ macrophage subpopulation by transcriptional profiling.
(A) Post-sort analysis of CD45+CD11b+F4/80+Lyve-1– and CD45+CD11b+F4/80+Lyve-1+ cells that were submitted for bulk RNA-seq analysis. (B) scRNA-seq data showing genes associated with macrophages …
Figure 3 with 1 supplement
F4/80+Lyve-1+ cells are enriched for CD206 expression.
(A) Violin plot of Mrc1 expression in the single-cell RNA-seq dataset shown in Figure 2. (B) Mammary glands from 10-week-old mice (n = 4) were assessed for CD206 expression in CD45+CD11b+F4/80+Lyve-1…
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Figure 3—source data 1
Source data for graphs in panels B, F, and G.
- https://cdn.elifesciences.org/articles/57438/elife-57438-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
F4/80+Lyve-1+ cells are enriched for CD206 expression.
(A) Gating of flow cytometry for Lyve-1 expression in CD45+CD11b+F4/80+CD206– and CD45+CD11b+F4/80+CD206+ cells. (B) Gating of flow cytometry for CD206 expression in CD45+CD11b+F4/80+Lyve-1– and …
Figure 4
Lyve-1+ macrophages exhibit low turnover and self-renewal in the mammary gland.
(A) Schematic of shielded bone marrow chimera experiment. (B) Representative flow cytometry plots showing the gating and identification of host (CD45.2) and donor (CD45.1) cells in the blood and …
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Figure 4—source data 1
Source data for graphs in panels B and C.
- https://cdn.elifesciences.org/articles/57438/elife-57438-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Lyve-1+ cells localize to HA-enriched ECM in the mammary gland.
(A) Mammary glands from 6-week-old mice were immunostained for HA using HA binding protein (HABP, green) and smooth muscle actin (SMA, red) (n = 3, three images/sample). (B) Mammary glands from …
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Figure 5—source data 1
Source data for graph in panel C.
- https://cdn.elifesciences.org/articles/57438/elife-57438-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Lyve-1+ cells localize to HA-enriched ECM in the mammary gland.
Mammary glands were stained for F4/80, Lyve-1 and HABP to confirm that Lyve-1+ cells associated with HA are also F4/80+. Insets show higher magnification.
Figure 6 with 1 supplement
Macrophage depletion impacts ECM in the stroma.
(A) 5-week-old female mice were treated with pexidartinib for 2 weeks and mammary glands were harvested for analysis of macrophages for CD45+CD11b+F4/80+ by flow cytometry. Flow cytometry …
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Figure 6—source data 1
Source data for graphs in panels A, C, and D.
- https://cdn.elifesciences.org/articles/57438/elife-57438-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Macrophage depletion does not impact ductal elongation of branching.
Quantification of ductal elongation and branching in mammary glands from mice treated with either solvent or pexidartinib for 2 weeks. Ductal Branching DMSO vs pexidartinib, p=0.1081; Ductal Length …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, female) | FVB/N | Envigo | FVB/NHsd | |
| Strain, strain background (Mus musculus, female) | Balb/c CD45.2 Mice | Jackson Laboratories | Stock No.: 000651 | Balb/cJ Mouse Strain |
| Strain, strain background (Mus musculus) | Balb/c CD45.1 Mice | Jackson Laboratories | Stock No.: 006584 | CByJ.SJL(B6)-Ptprca/J Mouse Strain |
| Antibody | violetFluor 450-Conjugated anti-CD45.1 (mouse monoclonal) | Tonbo Biosciences | Cat #: 75–0453 U025 RRID:AB_2621949 | Flow cytometry: 1:200 |
| Antibody | Alexa Fluor 700-Conjugated anti-CD45.2 (mouse monoclonal) | BioLegend | Cat #: 109822 RRID:AB_493731 | Flow cytometry: 1:200 |
| Antibody | BUV395-conjugated anti-Ly6G (rat monoclonal) | BioLegend | Cat #: 563978 RRID:AB_2716852 | Flow cytometry: 1:400 |
| Antibody | Brilliant Violet 421-Conjugated Anti-CD64 (mouse monoclonal) | BioLegend | Cat #: 139309 RRID:AB_2562694 | Flow cytometry: 1:100 |
| Antibody | Brilliant violet 711-conjugated anti-CD11c (Armenian hamster monoclonal) | BioLegend | Cat #: 117349 RRID:AB_2563905 | Flow cytometry: 1:200 |
| Antibody | PE/Cy7-conjugated anti-CD11b (rat monoclonal) | BioLegend | Cat #: 101216 RRID:AB_312799 | Flow cytometry: 1:200 |
| Antibody | PE-conjugated anti-F4/80 (rat monoclonal) | BioLegend | Cat #: 123109 RRID:AB_893498 | Flow cytometry: 1:200 |
| Antibody | Biotinylated anti-Lyve-1 (goat polyclonal) | R and D Systems | Cat #: BAF2125 RRID:AB_2138529 | Flow cytometry:4 μg/mL |
| Antibody | Anti-CD16/CD32 (rat monoclonal) | eBioscience | Cat #: 14-0161-82 RRID:AB_467133 | Flow cytometry: 1:200 |
| Antibody | Rat anti-mouse monoclonal F4/80 (clone: CI: A3-1) | Bio-Rad laboratories | Cat# MCA497GA RRID:AB_323806 | IF 1:100 |
| Antibody | Goat polyclonal anti-mouse LYVE-1 (Ala24-Thr234) | R and D Systems | Cat# AF2125 RRID:AB_2297188 | IF 1:60 |
| Antibody | Anti-mannose receptor (rabbit polyclonal) | Abcam | Cat# ab64693 RRID:AB_1523910 | IF 1:1000 |
| Antibody | Anti-BrdU antibody [BU1/75 (ICR1)] | Abcam | Cat# ab6326 RRID:AB_305426 | IF 1:200 |
| Antibody | Hyaluronic acid bindingprotein, biotinylated | Millipore Sigma | Cat# 385911–50 UG | IF 1:100 |
| Antibody | Actin-smooth muscle (rabbit polyclonal) | Spring Bioscience | Cat# E2464 RRID:AB_95752 | IF 1:50 |
| Antibody | CD68 monoclonal antibody (514H12) | Thermo- Fisher Scientific | Cat# MA1-80133 RRID:AB_929283 | IF 1:100 |
| Antibody | Human LYVE-1 antibody (goat polyclonal) | R and D Systems | Cat# AF2089 RRID:AB_355144 | IF 1:60 |
| Antibody | Alexa 594 secondary antibody (donkey anti-mouse) | Thermo- Fisher Scientific | Cat# A-32744 RRID:AB_2762826 | IF 1:400 |
| Antibody | Alexa 594 secondary antibody (donkey anti-rat) | Thermo- Fisher Scientific | Cat# A-21209 RRID:AB_2535795 | IF 1:400 |
| Antibody | Alexa Fluor 488 conjugated streptavidin secondary antibody | Thermo- Fisher Scientific | Cat# S11223 | IF 1:400 |
| Antibody | Alexa 488 secondary antibody (donkey anti-goat) | Thermo- Fisher Scientific | Cat# A-11055 RRID:AB_2534102 | IF 1:400 |
| Antibody | Alexa 488 secondary antibody (goat anti-rabbit) | Thermo- Fisher Scientific | Cat# A-11008 RRID:AB_143165 | IF 1:400 |
| Antibody | Alexa 594 secondary antibody (goat anti-rat) | Thermo- Fisher Scientific | Cat # A-11007 RRID:AB_10561522 | IF 1:400 |
| Antibody | Alexa 594 secondary antibody (goat anti-rabbit) | Thermo- Fisher Scientific | Cat# A-11012 RRID:AB_2534079 | IF 1:400 |
| Antibody | Alexa Fluor 647 conjugated streptavidin secondary antibody | Thermo- Fisher Scientific | Cat# S-32357 | IF 1:400 |
| Commercial assay or kit | Hyaluronan enzyme-linked immunosorbent assay | Echelon Biosciences | Cat #: K-1200 | |
| Chemical compound, drug | Busulfan | Sigma-Aldrich | Cat #: B2635-10G | |
| Chemical compound, drug | Pexidartinib | Selleck Chem | Cat #: S7818 | |
| Chemical compound, drug | Heparin | Sigma-Aldrich | Cat #: H3149-10KU | |
| Chemical compound, drug | Collagenase A | Sigma-Aldrich | Cat #: 10103586001 | |
| Chemical compound, drug | DNase I | Sigma-Aldrich | Cat #: DN25-100MG | |
| Software, algorithm | GraphPad Prism 8.0 | GraphPad Prism 8.0 | RRID:SCR_002798 | http://www.graphpad.com/scientific-software/prism/ |
| Software, algorithm | ImageJ 2.0.0 | ImageJ 2.0.0 | RRID:SCR_003070 | https://imagej.nih.gov/ij/download.html |
| Software, algorithm | Hisat2 | Kim et al., 2015 | RRID:SCR_015530 | version 2.0.2 |
| Software, algorithm | DESeq2 software | Love et al., 2014 | RRID:SCR_015687 | version 1.20.0 |
| Software, algorithm | ggplot2 | R | RRID:SCR_014601 | v3.0.0 |
| Software, algorithm | pheatmap | R | RRID:SCR_016418 | 1.0.12 |
| Other | Flow Cytometry Perm Buffer (10X) | Tonbo Biosciences | Cat #: TNB-1213-L150 | |
| Other | Fixable Viability Dye eFluor 780 | eBioscience | Cat #: 65-0865-14 | Flow cytometry: 1:1000 dilution |
| Other | APC-conjugated streptavidin | eBioscience | Cat #: 17-4317-82 | Flow cytometry: 1:100 dilution |
| Other | ProLong Gold Antifade Mountant (DAPI) | Thermo- Fisher Scientific | Cat# P36931 | |
| Other | Trichrome stain kit | Abcam | Cat# ab150686 | |
| Other | Antigen Retrieval Buffer (100X EDTA Buffer, pH 8.0) | Abcam | Cat# ab93680 | 1x |
| Other | Antigen unmasking solution, PH 6.0 | Thermo- Fisher Scientific | Cat# NC9401067 | 1x |
Additional files
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Supplementary file 1
List of 155 differentially expressed genes in CD45+CD11b+F4/80+Lyve-1– and CD45+CD11b+F4/80+Lyve-1+ bulk RNA-seq analysis.
- https://cdn.elifesciences.org/articles/57438/elife-57438-supp1-v2.xlsx
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Supplementary file 2
List of the top 100 differentially regulated genes in each cluster from the scRNA-seq analysis.
- https://cdn.elifesciences.org/articles/57438/elife-57438-supp2-v2.xlsx
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Supplementary file 3
GSEA analysis using gene sets created from a differentially expressed ranked list from the bulk RNA-seq data (adjusted p-value <0.1 and a fold-change >0.25) to determine enrichment of cluster 4.
- https://cdn.elifesciences.org/articles/57438/elife-57438-supp3-v2.xlsx
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Supplementary file 4
List of top gene sets identified by GSEA analysis including core enriched genes.
- https://cdn.elifesciences.org/articles/57438/elife-57438-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57438/elife-57438-transrepform-v2.docx
