While macrophages are among the most abundant immune cell type found within primary and metastatic mammary tumors, how their complexity and heterogeneity change with metastatic progression remains unknown. Here, macrophages were isolated from the lungs of mice bearing orthotopic mammary tumors for single-cell RNA sequencing (scRNA-seq). Seven distinct macrophage clusters were identified, including populations exhibiting enhanced differential expression of genes related to antigen presentation (H2-Aa, Cd74), cell cycle (Stmn1, Cdk1), and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrated a profile concordant with lipid-associated macrophages (Lgals3, Trem2). Compared with nontumor-bearing controls, the number of these cells per gram of tissue was significantly increased in lungs from tumor-bearing mice, with the vast majority costaining positively with the alveolar macrophage marker Siglec-F. Enrichment of genes implicated in pathways related to lipid metabolism as well extracellular matrix remodeling and immunosuppression was observed. In addition, these cells displayed reduced capacity for phagocytosis. Collectively, these findings highlight the diversity of macrophages present within metastatic lesions and characterize a lipid-associated macrophage subset previously unidentified in lung metastases. Significance: scRNA-seq of macrophages isolated from lung metastases reveals extensive macrophage heterogeneity and identifies a novel subpopulation enriched for genes involved in lipid metabolism, extracellular matrix remodeling, and immunosuppression.
Atherosclerotic plaque formation is driven by the continued expansion of cholesterol loaded foamy macrophages within the arterial intima. Foamy macrophages are primarily derived from newly recruited monocytes, but factors regulating monocyte specification toward foamy macrophage differentiation and prolonged survival in plaque remain poorly understood. We used trajectory analysis of integrated single cell RNA-seq data, along with a genome-wide CRISPR screening approach to identify Triggering Receptor Expressed on Myeloid Cells 2 (Trem2) as a candidate regulator for foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up additional oxidized low density lipoprotein (LDL) cholesterol in vitro. Competitive chimera experiments showed that Trem2-deficient macrophages were less competent to form foamy macrophages when competed against Trem2-sufficient macrophages in vivo. In addition, myeloid specific conditional deletion of Trem2 resulted in a dramatic attenuation of plaque progression, even when targeted in established atherosclerotic lesions. This was independent of changes in circulating inflammatory cytokines, monocyte recruitment, or serum cholesterol levels, but due to a reduction in plaque macrophage proliferation and enhanced cell death. Mechanistically, we link Trem2-deficient macrophages with an inability for cells to sense cholesterol loading and failure to upregulate efflux molecules. Accumulation of cholesterol in the endoplasmic reticulum enhanced activation of the ER-stress response that increased susceptibility for cholesterol-toxicity and cell death in foamy Trem2-deficient macrophages. Overall, this study identifies Trem2 as a regulator of foamy macrophage differentiation, atherosclerotic plaque growth, and as a putative therapeutic target for future intervention studies.
Arterial stiffness is an important biomarker for many cardiovascular diseases. Shear wave elastography is a recent technique aimed at estimating local arterial stiffness using guided wave inversion (GWI), i.e. matching the computed and measured wave dispersion. This paper develops and validates a new GWI approach by synthesizing various recent observations and algorithms: (a) refinements to signal processing to obtain more accurate experimental dispersion curves; (b) an efficient forward model to compute theoretical dispersion curves for immersed, incompressible cylindrical waveguides; (c) an optimization framework based on the recent observation that the measured dispersion curve is multimodal, i.e. it matches for not one but two different wave modes in two different frequency ranges. The resulting inversion approach is validated using extensive experimental data from rubber tube phantoms, not only for modulus estimation but also to simultaneously estimate modulus and wall thickness. The observations indicate that the modulus estimates are best performed with the information on wall thickness. The approach, which takes less than half a minute to run, is shown to be accurate, with the modulus estimated with less than 4% error for 70% of the experiments.
Tissue‐resident macrophages are present in all tissues where they perform homeostatic and immune surveillance functions. In many tissues, resident macrophages develop from embryonic progenitors, which mature into a self‐maintaining population through local proliferation. However, tissue‐resident macrophages can be supported by recruited monocyte‐derived macrophages during scenarios such as tissue growth, infection, or sterile inflammation. Circulating blood monocytes arise from hematopoietic stem cell progenitors and possess unique gene profiles that support additional functions within the tissue. Determining cell origins (ontogeny) and cellular turnover within tissues has become important to understanding monocyte and macrophage contributions to tissue homeostasis and disease. Fate mapping, or lineage tracing, is a promising approach to tracking cells based on unique gene expression driving reporter systems, often downstream of a Cre‐recombinase–mediated excision event, to express a fluorescent protein. This approach is typically deployed temporally with developmental stage, disease onset, or in association with key stages of inflammation resolution. Importantly, myeloid fate mapping can be combined with many emerging technologies, including single‐cell RNA‐sequencing and spatial imaging. The application of myeloid cell fate mapping approaches has allowed for impactful discoveries regarding myeloid ontogeny, tissue residency, and monocyte fate within disease models. This protocol outline will discuss a variety of myeloid fate mapping approaches, including constitutive and inducible labeling approaches in adult and embryo tissues. This article outlines basic approaches and models used in mice for fate mapping macrophages. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Adult Fate Mapping Basic Protocol 2: Embryonic Fate Mapping
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