Intracellular Iron Chelation Modulates the Macrophage Iron Phenotype with Consequences on Tumor Progression

A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S₎₂ shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.     

Alternative activation of tumor-associated macrophages by IL-4

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression, and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ macrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.     

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.     

Alternative activation of tumor-associated macrophages by IL-4: Priming for protumoral functions

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ acrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.Key words: interleukin-4, tumor-associated macrophage, tumor microenvironment, cytokines, cathepsin proteases     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

TAMs在贲门癌和非贲门癌组织中的表达及其临床意义冰

目的：观察不同部位胃癌（贲门癌与非贲门癌）组织中肿瘤相关巨噬细胞（tumor associated macrohags,TAMs）的表达及其与血管生成的关系,并进一步分析其与临床病理特征的相关性。方法：利用免疫组化方法标记TAMs（抗CD68抗体）和血管内皮细胞（抗CD34抗体）,检测90例胃癌中（包括18例贲门癌与72例非贲门癌）TAMs、微血管密度（microvesseldensity,MVD）的表达情况。结果：①TAMs与胃癌的部位、分化程度、浸润深度及临床分期呈显著正相关（P均〈0．05）;②MVD与胃癌的部位和分化程度呈显著正相关（P〈0．05）;结论：TAM可以促进胃癌的发展,这可能与TAM促进肿瘤的血管生成有关;TAM的表达可能是影响贲门癌与非贲门癌生物学行为差异的因素之一。     

CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.     

胃癌中TAMs与血管生成及转移相关性的分析

目的：研究胃癌中肿瘤相关巨噬细胞（tumor associated macrophages,TAMs）的表达及其与肿瘤血管生成的关系,进而分析其与转移的相关性。方法：利用免疫组化双染方法同时标记TAMs（抗CD68抗体）和内皮细胞（抗CD34抗体）,检测90例胃癌中TAMs、微血管密度（microvessel density,MVD）。结果：①TAMs与胃癌的大小、淋巴结转移、远处转移呈显著正相关（P均〈0.05）;②MVD与胃癌的远处转移呈显著正相关（P均〈0.05）;③在胃癌组织中TAMs与MVD的表达呈显著的正相关（r=0.325,P=0.002）。结论：TAMs可能通过促进胃癌血管的生成,进而促进胃癌细胞发生血管内渗,最终实现胃癌细胞的转移。     

Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment

Background

Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue.

Results

We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells.

Conclusions

We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

     

In Vitro Generation of Monocyte-Derived Macrophages under Serum-Free Conditions Improves Their Tumor Promoting Functions

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.     

Anti-tumor effect of aquaporin 3 monoclonal antibody on syngeneic mouse tumor model

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun.Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs.These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.     

Absent in melanoma 2-mediating M1 macrophages facilitate tumor rejection in renal carcinoma

Absent in melanoma 2 (AIM2) as an immune regulator for the regulation of tumor-associated macrophages (TAMs) function is unclear in tumor development. Here, the AIM2 function was investigated in TAMs-mediated malignant behaviors of renal carcinoma. The correlation analysis result showed that the AIM2 expression in TAMs was negatively correlated with the percentages of M2-like polarization phenotype in human or murine renal cancer specimens. By the cocultured assay with bone marrow-derived macrophages (BMDMs) and Renca cells, overexpression of AIM2 in macrophages enhanced the inflammasome activation and reversed the phenotype from M2 to M1. Compared with BMDMs-Ctrl cocultured group, BMDMs-AIM2 cocultured group showed reduced tumor cell proliferation and migration. The blockade of inflammasome activation by the inhibitor Ac-YVAD-CMK abrogated AIM2-mediated M1 polarization and the inhibition of tumor cell growth. To evaluate the therapeutic efficacy of AIM2-mediated M1 macrophages in vivo, BMDMs-AIM2 were intravenously injected into subcutaneous Renca-tumor mice. The results showed that the infiltration of M1 TAMs was increased and tumor growth was suppressed in BMDMs-AIM2-treated mice when compared with BMDMs-Ctrl-treat mice. Accordingly, the blockade of inflammasome activation reduced the anti-tumor activities of BMDMs-AIM2. Moreover, the lung metastases of renal carcinoma were suppressed by the administration of BMDMs-AIM2 accompanied with the reduced tumor foci. These results demonstrated that AIM2 enhanced TAMs polarization switch from anti-inflammatory M2 phenotypy to pro-inflammatory M1 through inflammasome signaling activation, thus exerting therapeutic intervention in renal carcinoma models. Our results provide a possible molecular mechanism for the modulation of TAMs polarization in tumor microenvironment and open a new potential therapeutic approach for renal cancer.     

miR-125a/b inhibits tumor-associated macrophages mediated in cancer stem cells of hepatocellular carcinoma by targeting CD90

Cancer stem cells promote tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, emerging evidence indicates tumor-associated macrophages (TAMs) play an important role in tumor progression. However, TAMs often occurs with unknown mechanisms. As an important mediator in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins, which involves tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate stem cell properties in HCC. TAMs were isolated from the tissues of HCC. microRNA (miRNA) expression profiles of TAMs were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate the crosstalk between TAMs and tumor cells mediated by TAMs exosomes. In this study, we showed that TAMs exosomes promote HCC cell proliferation and stem cell properties. Using miRNA profiles assay, we identified significantly lower levels of miR-125a and miR-125b in exosomes and cell lysate isolated from TAMs. Functional studies revealed that the HCC cells were treated with TAM exosomes or transfected with miR-125a/b suppressed cell proliferation and stem cell properties by targeting CD90, a stem cell marker of HCC stem cells. The study indicated that miR-125a/b targeting CD90 played important roles in cancer stem cells of HCC.     

Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.     

Proangiogenic TIE2+/CD31+ macrophages are the predominant population of tumor-associated macrophages infiltrating metastatic lymph nodes

Tumor-associated macrophages (TAMs) accumulate in various cancers and promote tumor angiogenesis and metastasis, and thus may be ideal targets for the clinical diagnosis of tumor metastasis with high specificity. However, there are few specific markers to distinguish between TAMs and normal or inflammatory macrophages. Here, we show that TAMs localize in green fluorescent protein-labeled tumors of metastatic lymph nodes (MLNs) from B16F1 melanoma cells but not in necrotic tumor regions, suggesting that TAMs may promote the growth of tumor cells and the progression of tumor metastasis. Furthermore, we isolated pure populations of TAMs from MLNs and characterized their gene expression signatures compared to peritoneal macrophages (PMs), and found that TAMs significantly overexpress immunosuppressive cytokines such as IL-4, IL-10, and TGF-β as well as proangiogenic factors such as VEGF, TIE2, and CD31. Notably, immunological analysis revealed that TIE2⁺/CD31⁺ macrophages constitute the predominant population of TAMs that infiltrate MLNs, distinct from tissue or inflammatory macrophages. Importantly, these TIE2⁺/CD31⁺ macrophages also heavily infiltrated MLNs from human breast cancer biopsies but not reactive hyperplastic LNs. Thus, TIE2⁺/CD31⁺ macrophages may be a unique histopathological biomarker for detecting metastasis in clinical diagnosis, and a novel and promising target for TAM-specific cancer therapy.     

Tumor-associated macrophages: Effectors of angiogenesis and tumor progression

Tumor-associated macrophages (TAMs) are a prominent inflammatory cell population in many tumor types residing in both perivascular and avascular, hypoxic regions of these tissues. Analysis of TAMs in human tumor biopsies has shown that they express a variety of tumor-promoting factors and evidence from transgenic murine tumor models has provided unequivocal evidence for the importance of these cells in driving angiogenesis, lymphangiogenesis, immunosuppression, and metastasis. This review will summarize the mechanisms by which monocytes are recruited into tumors, their myriad, tumor-promoting functions within tumors, and the influence of the tumor microenvironment in driving these activities. We also discuss recent attempts to both target/destroy TAMs and exploit them as delivery vehicles for anti-cancer gene therapy.