Tumor-associated Macrophages In Breast Cancer Research Articles

AXL/GAS6 signaling governs differentiation of tumor-associated macrophages in breast cancer

Most epithelial cancers are infiltrated by prognostically relevant myelomonocytic cells. Immunosuppressive tumor associated macrophages (TAMs) and their precursor monocytic-MDSCs have previously been associated with worse outcomes in human breast cancer (BCa), yet the mechanism of immunosuppressive TAMs-polarization from myelomonocytic precursors is not completely understood. In this study, we show that persuaded AXL/GAS6 pathway alters macrophage phenotype from HLA-DRhighCD206low CD163low classical phagocytic into HLA-DRlowCD206highCD163high immunosuppressive ones with accelerated BCa progression, and increased angiogenesis signature and invasion ability of cancer cells at tumor beds. Notably, both AXL and GAS6 expressions are upregulated in human invasive breast carcinoma, with maximum expression in triple negative histology type. Mechanistically, we demonstrate that AXL/GAS6 signaling drives immunosuppression by governing increased immunosuppressive IL10 production while dampening IL-1β expression within the tumor microenvironment (TME) of BCa. Further, AXL/GAS6 signaling promotes angiogenesis through the activation of PI3K/AKT and NF-κB signaling pathways. Our results unveil role of AXL/GAS6 axis in the differentiation of TAMs, which governs malignant growth, and suggest that therapies that uncouple AXL/GAS6 axis may exhibit therapeutic opportunity for otherwise undruggable Triple Negative Breast Cancer (TNBC) patients.

Single-cell transcriptional atlas of tumor-associated macrophages in breast cancer

BackgroundThe internal heterogeneity of breast cancer, notably the tumor microenvironment (TME) consisting of malignant and non-malignant cells, has been extensively explored in recent years. The cells in this complex cellular ecosystem activate or suppress tumor immunity through phenotypic changes, secretion of metabolites and cell-cell communication networks. Macrophages, as the most abundant immune cells within the TME, are recruited by malignant cells and undergo phenotypic remodeling. Tumor-associated macrophages (TAMs) exhibit a variety of subtypes and functions, playing significant roles in impacting tumor immunity. However, their precise subtype delineation and specific function remain inadequately defined.MethodsThe publicly available single-cell transcriptomes of 49,141 cells from eight breast cancer patients with different molecular subtypes and stages were incorporated into our study. Unsupervised clustering and manual cell annotation were employed to accurately classify TAM subtypes. We then conducted functional analysis and constructed a developmental trajectory for TAM subtypes. Subsequently, the roles of TAM subtypes in cell-cell communication networks within the TME were explored using endothelial cells (ECs) and T cells as key nodes. Finally, analyses were repeated in another independent publish scRNA datasets to validate our findings for TAM characterization.ResultsTAMs are accurately classified into 7 subtypes, displaying anti-tumor or pro-tumor roles. For the first time, we identified a new TAM subtype capable of proliferation and expansion in breast cancer-TUBA1B+ TAMs playing a crucial role in TAMs diversity and tumor progression. The developmental trajectory illustrates how TAMs are remodeled within the TME and undergo phenotypic and functional changes, with TUBA1B+ TAMs at the initial point. Notably, the predominant TAM subtypes varied across different molecular subtypes and stages of breast cancer. Additionally, our research on cell-cell communication networks shows that TAMs exert effects by directly modulating intrinsic immunity, indirectly regulating adaptive immunity through T cells, as well as influencing tumor angiogenesis and lymphangiogenesis through ECs.ConclusionsOur study establishes a precise single-cell atlas of breast cancer TAMs, shedding light on their multifaceted roles in tumor biology and providing resources for targeting TAMs in breast cancer immunotherapy.

Comprehensive Molecular Analyses of an M2-Like Tumor-Associated Macrophage for Predicting the Prognosis and Immunotherapy in Breast Cancer.

The involvement of M2-like tumor-associated macrophages (TAMs) in the advancement and treatment of cancer has been widely documented. This study aimed to develop a new signature associated with M2-like TAMs to predict the prognosis and treatment response in individuals diagnosed with breast cancer (BC). Weighted gene co-expression network analysis (WGCNA) was used to identity for M2-like TAM-related modular genes. The M2-like TAM-related modular subtype was identified using unsupervised clustering. WGCNA identified 722 M2-like TAM genes, 204 of which were associated with recurrence-free survival (RFS). Patients in cluster 1 exhibited upregulated cancer-related pathways, a higher proportion of triple-negative breast cancer (TNBC) subtypes, lower expression of immune checkpoints, and worse prognosis. Cluster 2 was characterized by upregulated immune-related pathways, a higher proportion of luminal A subtypes, and higher expression of immune checkpoints. A prognostic signature was created and confirmed using an independent dataset. A well-built nomogram can accurately forecast the survival outcomes for every individual. Furthermore, patients classified as low-risk exhibited a more favorable outlook, elevated tumor microenvironment (TME) score, and superior reaction to immunotherapy. In conclusion, we discovered 2 different types of M2-like TAMs and developed a prognostic signature revealing the diversity of M2-like TAMs in BC and their correlation with immune status and prognosis. This feature can predict the prognosis and immunotherapeutic effects of BC and offer novel concepts and approaches for tailoring BC treatment.

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

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. 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. 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. 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.

Crosstalk Between Macrophages and Breast Cancer Cells: Networking Within Tumors.

Tumor associated macrophages (TAMs) are one of the most prominent immune cells in the breast tumor microenvironment (TME). TAMs are categorised into classically activated anti-tumorigenic M1 and alternatively activated pro-tumorigenic M2 macrophages. TAMs are known to promote cancer pathogenesis by facilitating cancer cell and cancer stem cell growth, angiogenesis, immune evasion, invasion, and migration. Consequently, TAMs drive cancer progression towards metastasis. This chapter describes the role of TME in driving monocyte recruitment and polarization toward the M2 phenotype. We also illustrate the modalities of intercellular networking such as paracrine signaling, exosomes, and tunneling nanotubes (TNTs) that TAMs and cancer cells employ within TME to communicate with each other and with other cells of TME to facilitate the dynamic process of cancer progression. Finally, we discuss the clinical implications of TAMs in breast cancer and potential therapeutic strategies targeting TAM recruitment, polarization, and TAM-mediated immune evasion for effective cancer therapy.

Multifunctional nanoparticles inhibit tumor and tumor-associated macrophages for triple-negative breast cancer therapy

HypothesisTumor-associated macrophages (TAM) are the mainstay of immunosuppressive cells in the tumor microenvironment, and elimination of M2-type macrophages (M2-TAM) is considered as a potential immunotherapy. However, the interaction of breast cancer cells with macrophages hinders the effectiveness of immunotherapy. In order to improve the efficacy of triple-negative breast cancer (TNBC) therapy, strategies that simultaneously target the elimination of M2-TAM and breast cancer cells may be able to achieve a better therapy. ExperimentsLyP-SA/AgNP@Dox multifunctional nanoparticles were synthesized by electrostatic adsorption. They were characterized by particle size, potential and spectroscopy. And the efficacy of multifunctional nanoparticles was evaluated in 4 T1 cell lines and M2 macrophages, including their cell uptake intracellular reactive oxygen species (ROS) production and the therapeutic effect. Furthermore, based on the orthotopic xenotransplantation model of triple negative breast cancer, the biological distribution, fluorescence imaging, biosafety evaluation and combined efficacy evaluation of the nanoplatform were performed. FindingsWe have successfully prepared LyP-SA/AgNP@Dox and characterized. Administering the nanosystem to 4 T1 tumor cells or M2 macrophages in culture induced accumulation of reactive oxygen species, destruction of mitochondria and apoptosis, and inhibited replication and transcription. Animal experiments demonstrated the nanoparticle had favorable targeting and antitumor activity. Our nanosystem may be useful for simultaneously inhibiting tumor and tumor-associated macrophages in breast cancer and, potentially, other malignancies.

Antitumor therapy for breast cancer: Focus on tumor-associated macrophages and nanosized drug delivery systems.

In breast cancer (BC), tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment and are closely related to poor prognosis. A growing number of studies have focused on the role of TAMs in BC progression and therapeutic strategies targeting TAMs. As an emerging treatment, the application of nanosized drug delivery systems (NDDSs) in the treatment of BC by targeting TAMs has attracted much attention. This review is to summarize the characteristics and treatment strategies targeting TAMs in BC and to clarify the applications of NDDSs targeting TAMs in the treatment of BC by targeting TAMs. The existing results related to characteristics of TAMs in BC, BC treatment strategies by targeting TAMs, and the applications of NDDSs in these strategies are described. Through analyzing these results, the advantages and disadvantages of the treatment strategies using NDDSs are discussed, which could provide advices on designing NDDSs for BC treatment. TAMs are one of the most prominent noncancer cell types in BC. TAMs not only promote angiogenesis, tumor growth and metastasis but also lead to therapeutic resistance and immunosuppression. Mainly four strategies have been used to target TAMs for BC therapy, which include depleting macrophages, blocking recruitment, reprogramming to attain an anti-tumor phenotype, and increasing phagocytosis. Since NDDSs can efficiently deliver drugs to TAMs with low toxicity, they are promising approaches for targeting TAMs in tumor therapy. NDDSs with various structures can deliver immunotherapeutic agents and nucleic acid therapeutics to TAMs. In addition, NDDSs can realize combination therapies. TAMs play a critical role in the progression of BC. An increasing number of strategies have been proposed to regulate TAMs. Compared with free drugs, NDDSs targeting TAMs improve drug concentration, reduce toxicity and realize combination therapies. However, in order to achieve better therapeutic efficacy, there are still some disadvantages that need to be considered in the design of NDDSs. TAMs play an important role in the progression of BC, and targeting TAMs is a promising strategy for BC therapy. In particular, NDDSs targeting TAMs have unique advantages and are potential treatments for BC.

ODP543 Chemotherapy-induced extracellular HSP70 enhances pro-tumorigenic effects of macrophages in breast cancers.

Abstract Breast cancer is one of the most common types of malignancies in women worldwide. Cytotoxic chemotherapy has improved the prognosis of aggressive phenotype of breast cancer patients including triple-negative breast cancers. However, resistance to chemotherapy is an important problem. Tumor-associated macrophages (TAMs) are primary components of tumor microenvironment and promote tumor progression by releasing various cytokines. Pro-tumorigenic effects of TAMs are known to induce chemoresistance in breast cancers. On the other hand, we previously showed that pro-tumorigenic effects of TAMs were enhanced in breast cancer tissues following chemotherapy by immunohistochemical study. Here, we demonstrated that breast carcinoma cells exposed by chemotherapy induced pro-tumorigenic effects of TAMs. THP-1 human monocyte cell-derived macrophages were interacted with MDA-MB-231 and MDA-MB-453 human breast cancer cells treated with epirubicin (EPI), and conditioned media from them significantly promoted proliferation and migration of breast cancer cells. We found that the expression of heat shock protein (HSP) 70 in the cytoplasm of MDA-MB-231 cells was upregulated following EPI treatment. HSP70 is an intracellular molecular chaperone and recently described with extracellular actions in stressful conditions. However, extracellular HSP70 action on TAMs in breast cancer has remained largely unknown. MDA-MB-231 cells treated with EPI and EPI-resistant MDA-MB-231 subline promoted secretion of HSP70, especially exosomal HSP70. Then, HSP70 knockdown in MDA-MB-231 cells were performed using siRNA. Macrophages cultured with HSP70 suppressed conditioned media from MDA-MB-231 cells showed decreased expression of CD163, a marker of pro-tumorigenic macrophages. Furthermore, HSP70 suppressed conditioned media downregulated the expression of transforming growth factor (TGF)-beta and colony stimulating factor (CSF)-1 in MDA-MB-231 cells, which are known to increase pro-tumorigenic effects of macrophages. Then, we performed immunohistochemistry for HSP70 in 116 breast carcinoma tissues. HSP70 immunoreactivity in the cytoplasm of breast carcinoma cells was correlated with stage, lymph node metastasis, pathological T factor, histological grade, Ki-67 LI, and negatively correlated with ER and progesterone receptor (PR). Furthermore, immunoreactivity of HSP70 was increased in breast cancer patients who had received neoadjuvant chemotherapy. When we correlated HSP70 with clinical outcome, HSP70 immunoreactivity was correlated with increased risk of recurrences and breast-cancer-specific mortality. Importantly, macrophage infiltration was correlated with increased risk of recurrences only in the HSP70 positive group, but not in the HSP70-negative group, which indicates that HSP70 may be associated with the pro-tumorigenic effects of TAMs in breast cancers. Taken together, breast carcinoma cells promoted secretion of extracellular HSP70 following chemotherapy and it might induce chemoresistance by upregulating pro-tumorigenic effects of macrophages, directly or indirectly via regulation of cytokines expression in breast carcinoma cells. Extracellular HSP70 may be an important target to improve the prognosis of breast cancer patients. Presentation: No date and time listed

A cutting-edge immunomodulatory interlinkage between HOTAIR and MALAT1 in tumor-associated macrophages in breast cancer: A personalized immunotherapeutic approach.

Breast cancer (BC) is one of the most common cancers, accounting for 2.3 million cases worldwide. BC can be molecularly subclassified into luminal A, luminal B HER2-, luminal B HER2+, HER2+, and triple-negative breast cancer (TNBC). These molecular subtypes differ in their prognosis and treatment strategies; thus, understanding the tumor microenvironment (TME) of BC could lead to new potential treatment strategies. The TME hosts a population of cells that act as antitumorigenic such as tumor-associated eosinophils or pro-tumorigenic such as cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs), monocytic-derived populations such as MDSCs, or most importantly "tumor-associated macrophages (TAMs)," which are derived from CD14+ monocytes. TAMs are reported to have the pro-inflammatory phenotype M1, which is found only in the very early stages of tumor and is not correlated with progression; however, the M2 phenotype is anti-inflammatory that is correlated with tumor progression and metastasis. The current study focused on controlling the anti-inflammatory activity in TAMs of hormonal, HER2+, and TNBC by epigenetic fine-tuning of two immunomodulatory proteins, namely, CD80 and mesothelin (MSLN), which are known to be overexpressed in BC with pro-tumorigenic activity. Long non-coding RNAs are crucial key players in tumor progression whether acting as oncogenic or tumor suppressors. We focused on the regulatory role of MALAT1 and HOTAIR lncRNAs and their role in controlling the tumorigenic activity of TAMs. This study observed the impact of manipulation of MALAT1 and HOTAIR on the expression of both CD80 and MSLN in TAMs of BC. Moreover, we analyzed the interlinkage between HOTAIR and MALAT1 as regulators to one another in TAMs of BC. The current study reported an upstream regulatory effect of HOTAIR on MALAT1. Moreover, our results showed a promising use of MALAT1 and HOTAIR in regulating oncogenic immune-modulatory proteins MSLN and CD80 in TAMs of HER2+ and TNBC. The downregulation of MALAT1 and HOTAIR resulted in the upregulation of CD80 and MSLN, which indicates that they might have a cell-specific activity in TAMs. These data shed light on novel key players affecting the anti-inflammatory activity of TAMs as a possible therapeutic target in HER2+ and TNBC.

Role of Biological Mediators of Tumor-Associated Macrophages in Breast Cancer Progression.

Breast cancer (BRCA) has become the most common cancer worldwide. The tumor microenvironment (TME) in the breast exerts a crucial role in promoting BRCA initiation, progression, and metastasis. Tumor-associated macrophages (TAMs) are the primary component of tumor-infiltrating immune cells through biological mediators that convert TME into malignant tumors. Combinations of these biological mediators can promote tumor growth, metastasis, angiogenesis, and immune suppression and limit the anti-tumor activity of conventional chemotherapy and radiotherapy. The present study aimed to highlight the functions of several biological mediators in the breast thatgenerate TME into malignant tumors. Furthermore, this review offers a rationale for TAM-targeted therapy as a novel treatment strategy for BRCA. This review emphasizes TAM-associated biological mediators of TME, viz., cancer- associated fibroblasts, endothelial cells, adipocytes, tumor-derived exosomes, extracellular matrix, and other immune cells, which facilitate TME in malignant tumors. Evidence suggests that the increased infiltration of TAMs and elevated expression of TAMrelated genes are associated with a poor prognosis of BRCA. Based on these findings, TAM-targeted therapeutic strategies, including inhibitors of CSF-1/CSF-1R, CCL2/CCR2, CCL5-CCR5, bisphosphonate, nanoparticle, and exosomal-targeted delivery have been developed, and are currently being employed in intervention trials. This review concludes the roles of biological mediators of TME that interact with TAMs in BRCA, providing a rationale for TAM-targeted therapy as a novel treatment approach for BRCA.

Osteoclast-like stromal giant cells in breast cancer likely belong to the spectrum of immunosuppressive tumor-associated macrophages.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures. Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45–85%) and high TAMs (range, 35–75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues. Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.

CD14 macrophage and IL-10 levels in the peripheral blood of breast cancer patients and their diagnostic value*

Abstract Objective To explore the correlation between macrophages and interleukin-10 (IL-10 in the peripheral blood of breast cancer (BC) patients and the diagnostic value of joint detection. Methods BC patients (n = 50) and healthy controls (n = 40) were prospectively recruited. The percentage of circulating cluster of differentiation 14 (CD 14) macrophage cells was analyzed by flow cytometry, and an enzyme-linked immunosorbent assay (ELISA) was used to detect IL-10 expression levels. Receiver operating characteristic (ROC) curves were used to verify the diagnostic value of the models based on the expression of CD14 macrophage cell populations and IL-10. In addition, the association between model expression and clinicopathological characteristics was investigated. Another 30 patients with BC and 30 with benign breast disease were selected to validate the IL-10 and CD14 macrophage joint detection model using the same method. Results CD14 macrophage and IL-10 expression levels in BC patients were higher than those in healthy controls (P < 0.05). The ROC curve showed that the area under the curve (AUC) of CD14+ macrophages combined with IL-10 was 0.830, the sensitivity was 72.0%, and the specificity was 87.5%. Its diagnostic efficiency was better than all other single and joint detections. Correlation analysis of clinicopathological features showed that IL-10 and CD14+ macrophage joint detection was significantly correlated with tumor size, tumor-node-metastasis (TNM) stage, and lymph node, estrogen receptor (ER), and Ki-67 expression (P < 0.05). The validation analysis results were consistent with the test results. Conclusion Peripheral blood macrophages can be an independent diagnostic marker for BC. Joint detection of CD14- macrophages and IL-10 suggests poor prognosis, which has unlimited potential to guide BC development and provides a new theory for studying tumor-associated macrophages in BC.

RON (MST1R) and HGFL (MST1) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk.

Simple SummaryRON is a protein that sits on the surface of a cell and passes signals from the outside-in when encountering the HGFL protein. In cancer, the RON protein is often found at high levels leading to too many signals being passed. Given the close proximity of RON and HGFL in the human genome, we hypothesized that the two proteins are overproduced together in cancer cells. Other cells that interact with cancer cells, such as macrophages, also express RON and can respond to HGFL produced by cancer cells, creating a diverse response to overly abundant RON and HGFL. In this study, we evaluated how RON and HGFL in cancer cells affect cancer growth and progression and their response to other cells, and examined explanations for increases in RON and HGFL abundance in breast cancer.Background: Aberrant RON signaling is present in numerous cancers including breast cancer. Evidence suggests that the ligand, hepatocyte growth factor-like (HGFL), is also overexpressed in breast cancer. RON (MST1R) and HGFL (MST1) genes are located on human chromosome 3 and mouse chromosome 9 respectively and are found near each other in both species. Based on co-expression patterns, we posited that RON and HGFL are co-regulated and that coordinate upregulation drives aggressive tumorigenesis. Methods: Mouse models were used to establish the functional significance of RON and HGFL co-overexpression on the activation of tumor cells and tumor-associated macrophages in breast cancer. TCGA and METABRIC gene expression and alteration data were used to query the relationships between MST1R and MST1 in breast cancer. Results: In tumor models, physiologic sources of HGFL modestly improve Arginase-1+ (M2) macrophage recruitment to the tumor proper. Tumor-cell produced HGFL functions in autocrine to sustain tumor cell RON activation and MAPK-dependent secretion of chemotactic factors and in paracrine to activate RON on macrophages and to promote breast cancer stem cell self-renewal. In silico analyses support that RON and HGFL are co-expressed across virtually all cancer types including breast cancer and that common genomic alterations do not appear to be drivers of RON/HGFL co-overexpression. Conclusions: Co-overexpression of RON and HGFL in breast cancer cells (augmented by physiologic sources of HGFL) promotes tumorigenesis through autocrine-mediated RON activation/RON-dependent secretome changes and paracrine activation of macrophage RON to promote breast cancer stem cell self-renewal.

Extracellular Vesicles from M1-Polarized Macrophages Combined with Hyaluronic Acid and a β-Blocker Potentiate Doxorubicin's Antitumor Activity by Downregulating Tumor-Associated Macrophages in Breast Cancer.

One of the main reasons for cancer’s low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the β-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-κB was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-β. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer.

Isoforms of Neuropilin-2 Denote Unique Tumor-Associated Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis. Neuropilin-2 (NRP2), consisting of the NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality, with NRP2b promoting increased Akt signaling via receptor tyrosine kinases including VEGFRs, MET, and PDGFR. Although NRP2 has been shown to regulate macrophage/TAM biology, the role of the individual NRP2a/NRP2b isoforms in TAMs has yet to be evaluated. Using transcriptional profiling and spectral flow cytometry, we show that NRP2 isoform expression was significantly higher in TAMs from murine mammary tumors. NRP2a/NRP2b levels in human breast cancer metastasis were dependent upon the anatomic location of the tumor and significantly correlated with TAM infiltration in both primary and metastatic breast cancers. We define distinct phenotypes of NRP2 isoform-expressing TAMs in mouse models of breast cancer and within malignant pleural effusions from breast cancer patients which were exclusive of neuropilin-1 expression. Genetic depletion of either NRP2 isoform in macrophages resulted in a dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, depletion of NRP2b, but not NRP2a, inhibited production of IL-6. These results suggest that NRP2 isoforms regulate both shared and unique functionality in macrophages and are associated with distinct TAM subsets in breast cancer.

Tumor Properties Mediate the Relationship between Peripheral Blood Monocytes and Tumor-Associated Macrophages in Breast Cancer

In cancer patients, circulating monocytes show functional alterations. Since monocytes are precursors of tumor-associated macrophages (TAMs), TAMs ensuring tumor viability are potentially replenished through the recruitment of monocytes with specific properties. We demonstrated that locoregional metastasis and circulating factors, such as CD45-EpCAM + CD44 + CD24-/low circulating tumor cells, and serum MCP-1 and HMGB1 were statistically associated with modulation of the monocyte features in breast cancer patients. The count of circulating CD45-EpCAM + cells correlated with CD68+, CD163 + monocyte in blood, and with density of CD68 + TAM in breast cancer tumors. Overall, the relationship between monocytes and TAMs is mediated by the tumor in breast cancer patients.

Abstract ES11-2: Macro and Micro: Macrophage and microbiome diversity in the breast cancer microenvironment

Abstract The breast tumor microenvironment (TME), devoid of nutrients and oxygen, recruits tumor associated macrophages (TAMs) that promote angiogenesis, metastasis and tumor growth. Removal or conversion of TAMs to an anti-tumor phenotype enhances chemo- and immuno-therapy in pre-clinical models and establishes TAMs as targets for anti-cancer therapy. Translation to the clinic has been slow, likely because current strategies broadly target macrophages. Several subsets of TAMs have been identified, yet little is known about their subset diversity in breast cancer and response to therapy. The failure to characterize TAM subsets has likely hindered progress in therapeutic targeting. This educational talk will provide a broad overview of the diversity of TAMs as well as the microbiome and how they are potentially intertwined within the complex breast TME. The talk will incorporate the state of the field to include elucidating the complexity of TAMs in breast cancer including their ontogeny, tissue specificity, phenotype, subset diversity and spatial relationship within the breast tumor. In addition, how the tumor microbiome may influence myeloid function and phenotype will be explored. Citation Format: JL Guerriero. Macro and Micro: Macrophage and microbiome diversity in the breast cancer microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr ES11-2.

Targeted Imaging of Tumor Associated Macrophages in Breast Cancer

Abstract Breast cancer is a highly heterogeneous disease lacking prognostic markers. Tumor-associated macrophages (TAMs) in the tumor microenvironment are associated with distant metastasis as well as poorer outcomes in breast cancer. Therefore, monitoring TAMs may guide prognostic assessment. This study explores an imaging modality based on a two-step click chemistry procedure for detecting TAMs in breast cancer. Mannose-targeted liposomes (MAN-lipo-AAG) and non-targeted liposomes (lipo-AAG) encapsulating Ac4GalNAz were prepared and comprehensively characterized. The sizes of the prepared MAN-lipo-AAG and lipo-AAG were 126 ± 0.22 and 93 ± 0.23 nm, respectively. In vitro studies demonstrated higher uptake of MAN-lipo-AAG than lipo-AAG by RAW264.7 cells. Moreover, flow cytometry and confocal microscopy confirmed that bright, homogeneous fluorescence labeling was present on RAW264.7 cell membranes in the MAN-lipo-AAG group. Furthermore, in vivo analysis indicated that MAN-lipo-AAG, compared with lipo-AAG, had higher accumulation in a 4T1 xenograft model and higher uptake by mannose-overexpressing TAMs. This study describes a promising approach for specific and non-invasive TAM-targeted imaging via metabolic glycoengineering.

High grade breast ductal carcinomas have high density of tumor-associated macrophages.

The role of tumor-associated macrophages (TAMs) is double-natured and still controversial. Depending on different settings, macrophages may suppress or promote tumor growth. TAM density may be one of the predictive factors of treatment outcome in cancer patients. To evaluate the density of tumor-associated macrophages in breast cancer and its relationship with various histopathologic findings. 55patients with invasive ductal carcinoma of breast who underwent mastectomy were enrolled. Sections of tumor samples were stained and the density of CD68+ cells was evaluated. There was anassociation between estrogen receptor (ER) expression and CD68density (p = 0.010) as the higher densities of CD68were seen inER negative tumors. Moreover, there was a significant relationship between histological grade and CD68density (p =0.006). The higher TAM density is associated with higher tumor grade and negative ER expression in breast cancer tissues. These findings revealed that inflammation could have an important role in malignancies.

Tumor-Associated Macrophages: Critical Players in Drug Resistance of Breast Cancer.

Drug resistance is one of the most critical challenges in breast cancer (BC) treatment. The occurrence and development of drug resistance are closely related to the tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs), the most important immune cells in TIME, are essential for drug resistance in BC treatment. In this article, we summarize the effects of TAMs on the resistance of various drugs in endocrine therapy, chemotherapy, targeted therapy, and immunotherapy, and their underlying mechanisms. Based on the current overview of the key role of TAMs in drug resistance, we discuss the potential possibility for targeting TAMs to reduce drug resistance in BC treatment, By inhibiting the recruitment of TAMs, depleting the number of TAMs, regulating the polarization of TAMs and enhancing the phagocytosis of TAMs. Evidences in our review support it is important to develop novel therapeutic strategies to target TAMs in BC to overcome the treatment of resistance.