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

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. SIGNIFICANCE: 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.

Defining and targeting macrophage heterogeneity in the mammary gland and breast cancer.

INTRODUCTION: Macrophages are innate immune cells that are associated with extensive phenotypic and functional plasticity and contribute to normal development, tissue homeostasis, and diseases such as cancer. In this review, we discuss the heterogeneity of tissue resident macrophages in the normal mammary gland and tumor-associated macrophages in breast cancer. Tissue resident macrophages are required for mammary gland development, where they have been implicated in promoting extracellular matrix remodeling, apoptotic clearance, and cellular crosstalk. In the context of cancer, tumor-associated macrophages are key drivers of growth and metastasis via their ability to promote matrix remodeling, angiogenesis, lymphangiogenesis, and immunosuppression. METHOD: We identified and summarized studies in Pubmed that describe the phenotypic and functional heterogeneity of macrophages and the implications of targeting individual subsets, specifically in the context of mammary gland development and breast cancer. We also identified and summarized recent studies using single-cell RNA sequencing to identify and describe macrophage subsets in human breast cancer samples. RESULTS: Advances in single-cell RNA sequencing technologies have yielded nuances in macrophage heterogeneity, with numerous macrophage subsets identified in both the normal mammary gland and breast cancer tissue. Macrophage subsets contribute to mammary gland development and breast cancer progression in differing ways, and emerging studies highlight a role for spatial localization in modulating their phenotype and function. CONCLUSION: Understanding macrophage heterogeneity and the unique functions of each subset in both normal mammary gland development and breast cancer progression may lead to more promising targets for the treatment of breast cancer.

Characterization of Hyaluronan Localization in the Developing Mammary Gland and Mammary Tumors.

The extracellular matrix (ECM) is biochemically and biomechanically important for the structure and function of the mammary gland, which undergoes vast structural changes throughout pubertal and reproductive development. Although hyaluronan (HA) is a ubiquitous glycosaminoglycan (GAG) of the mammary gland ECM, extensive characterization of HA deposition in the mammary gland is lacking. Understanding physiologic HA metabolism is critical as this tightly controlled system is often hijacked in cancer. In the current studies, we characterize HA regulation throughout mammary gland development to better understand subsequent dysregulation of HA in mammary tumors. Using immunofluorescence (IF) imaging, we demonstrate that organized HA-rich septa exist in the mammary gland stroma throughout puberty, pregnancy, and involution. Furthermore, we find heterogeneous HA deposition within two murine models of breast cancer. Using cell specific isolation techniques, we characterize expression of genes associated with HA binding, synthesis, and degradation within EpCAM + epithelial cells, CD90.2 + fibroblasts, and F4/80 + macrophages isolated from mammary glands and tumors. Most notably, we identify elevated levels of the hyaluronidases Hyal1 and Hyal2 in tumor-association macrophages (TAMs), suggesting a role for TAM-mediated turnover of HA in the tumor microenvironment (TME). Gene expression is supported functionally by in vitro experiments in which macrophages treated with tumor-cell conditioned media exhibit increased hyaluronidase activity. These findings link TAMs to the direct degradation of HA within the TME of mammary tumors, which has negative implications for patient survival.