FIGURE 1 from LYVE-1–expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth

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posted on 2024-05-31, 14:20 authored by Alexis K. Elfstrum, Annisa H. Rumahorbo, Lyndsay E. Reese, Emma V. Nelson, Braedan M. McCluskey, Kathryn L. Schwertfeger

LYVE-1⁺ macrophages localize near stromal HA in the nulliparous mouse mammary gland and are associated with ECM regulation. A, Representative images from mammary gland capsular regions of 5-week Csf1r^fl/fl (top) and Lyve1^CreCsf1r^fl/fl (bottom) mice immunostained for LYVE-1 (green), CD206 (red), HABP (magenta), and DAPI (blue; n = 4 per genotype). B, Representative images from mammary gland stroma regions of 5-week Csf1r^fl/fl (top) and Lyve1^CreCsf1r^fl/fl (bottom) mice immunostained for LYVE-1 (green), F4/80 (red), HABP (magenta), and DAPI (blue; n = 4 per genotype). C, Mammary glands from 5-week Csf1r^fl/fl (gray) and Lyve1^CreCsf1r^fl/fl (blue) female mice were assessed for CD45⁻LYVE-1⁺ frequency by flow cytometry. D, Mammary glands from 5-week Csf1r^fl/fl (gray) and Lyve1^CreCsf1r^fl/fl (blue) female mice were assessed for CD45⁺F4/80⁺CD11b⁺ LYVE-1⁺ macrophage count by flow cytometry. Fold change of Lyve1 (E), Hyal1, and Hyal2 (F) RNA expression from 15-week Csf1r^fl/fl (gray) and Lyve1^CreCsf1r^fl/fl (blue) female mice. G, MFI of HABP in mammary glands assessing CD45⁺F4/80⁺CD11b⁺LYVE-1⁻ and CD45⁺F4/80⁺CD11b⁺LYVE-1⁺ macrophages. H, Mammary gland from 5-week and 15-week Csf1r^fl/fl (gray) and Lyve1^CreCsf1r^fl/fl (blue) female mice assessed for HA by ELISA and normalized by weight. I, Female mammary glands assessed by ECM qRT-PCR array with differentially downregulated genes associated with biological process Gene Ontology (GO) terms (black) and molecular function GO terms (white; n = 3 per genotype). *, P < 0.05; **, P < 0.01; ****, P < 0.0001, using Student t test. Scale bars, 100 µm. Each dot represents one mouse.

Funding

HHS | National Institutes of Health (NIH)

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ARTICLE ABSTRACT

Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peritumoral stroma, their functions within these regions are not well understood. Using a genetic mouse model of LYVE-1+ macrophage depletion, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that depletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single-cell RNA sequencing of macrophages isolated from these tumors reveals that depletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages toward a proinflammatory phenotype, as well as an increase in CD8+ T-cell infiltration. Together, these findings indicate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment. We have identified a macrophage subset in mouse mammary tumors associated with tumor structural components. When this macrophage subset is absent in tumors, we report a delay in tumor growth and an increase in antitumor immune cells. Understanding the functions of distinct macrophage subsets may allow for improved therapeutic strategies for patients with breast cancer.

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Keywords

Breast Cancer Immunology Tumor-associated macrophages Tumor Microenvironment Immune cells and the microenvironment

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