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      Human breast cancer cells educate macrophages toward the M2 activation status

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          Abstract

          Introduction

          The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression.

          Methods

          Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9).

          Results

          Clinically, we found that high numbers of CD163 + M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type ( p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype.

          Conclusions

          This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13058-015-0621-0) contains supplementary material, which is available to authorized users.

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          Most cited references36

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          Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.

          Angiogenesis is a key process in tumor growth and metastasis and is a major independent prognostic factor in breast cancer. A range of cytokines stimulate the tumor neovasculature, and tumor-associated macrophages have been shown recently to produce several important angiogenic factors. We have quantified macrophage infiltration using Chalkley count morphometry in a series of invasive breast carcinomas to investigate the relationship between tumor-associated macrophage infiltration and tumor angiogenesis, and prognosis. There was a significant positive correlation between high vascular grade and increased macrophage index (P = 0.03), and a strong relationship was observed between increased macrophage counts and reduced relapse-free survival (P = 0.006) and reduced overall survival (P = 0.004) as an independent prognostic variable. These data indicate a role for macrophages in angiogenesis and prognosis in breast cancer and that this cell type may represent an important target for immunoinhibitory therapy in breast cancer.
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            Polarization profiles of human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin.

            Monocytes/macrophages (MΦ), considered as plastic cells, can differentiate into either a pro-inflammatory (M1) subtype, also known as a classically activated subtype, or an anti-inflammatory alternatively activated subtype (M2) according to their microenvironment. Phenotypic markers of mouse polarized MΦ have been extensively studied, whereas their human counterparts remain less characterized. The main goal of this study was therefore to carefully characterize phenotypic and genomic markers of primary human MΦ generated from M-CSF-treated blood monocytes and polarized towards M1 or M2 subtype upon the action of lipopolysaccharide and interferon-γ (for M1) or interleukin (IL)-4 (for M2). Membrane expression of the markers CD80 and CD200R was found to be specific of human M1 and M2 polarized MΦ, respectively, whereas, by contrast, mannose receptor (CD206) expression did not discriminate between M1 and M2. mRNA expression analysis further identified six markers of M1 polarization (IL-12p35, CXCL10, CXCL11, CCL5, CCR7 and IDO1), five markers of M2 polarization (TGF-β, CCL14, CCL22, SR-B1 and PPARγ) and transcription factors involved in MΦ polarization. Ability of human M-CSF-generated MΦ to polarize toward M1 or M2 subtype was also associated with enhanced secretion of TNFα, IL-1β, IL-12p40, CXCL10 and IL-10 (for M1) or CCL22 (for M2). Moreover, the comparison of the expression of M1 markers in M-CSF- and GM-CSF-MΦ polarized towards M1 subtype has revealed similarities. In conclusion, we demonstrated that human M-CSF MΦ can polarize toward a M1 type after IFNγ/LPS stimulation. Moreover, the M1 and M2 markers of human polarized MΦ identified in the present study may be useful to better identify human MΦ subtypes, particularly at the tissue level, in order to better understand their respective roles in the development of pathologies. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Human macrophage polarization in vitro: maturation and activation methods compared.

              Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-β production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo. Copyright © 2014 Elsevier GmbH. All rights reserved.
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                Author and article information

                Contributors
                +358(0)503164101 , sofia.sousa@uef.fi
                regis.brion@univ-nantes.fr
                minnamaija.lintunen@utu.fi
                paukro@utu.fi
                jouko.sandholm@btk.fi
                jukka.monkkonen@uef.fi
                pirkko-liisa.kellokumpu-lehtinen@uta.fi
                susanna.lauttia@helsinki.fi
                olli.tynninen@hus.fi
                heikki.joensuu@hus.fi
                dominique.heymann@univ-nantes.fr
                jorma.maatta@utu.fi
                Journal
                Breast Cancer Res
                Breast Cancer Res
                Breast Cancer Research : BCR
                BioMed Central (London )
                1465-5411
                1465-542X
                5 August 2015
                5 August 2015
                2015
                : 17
                : 1
                : 101
                Affiliations
                [ ]School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, FI-70211 Kuopio, Finland
                [ ]INSERM, UMR957, Equipe LIGUE 2012, Nantes, F-44035 France
                [ ]Université de Nantes, Nantes atlantique universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, F-44035 France
                [ ]CHU de Nantes, Nantes, F-44035 France
                [ ]Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland
                [ ]Cell Imaging Core, Turku Centre for Biotechnology, University of Turku, and Åbo Akademi University, Turku, Finland
                [ ]Medical School, University of Tampere and Department of Oncology Tampere University Hospital, Tampere, Finland
                [ ]Laboratory of Molecular Oncology, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
                [ ]Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland
                [ ]Comprehensive Cancer Center, Helsinki University Hospital, and Department of Oncology, University of Helsinki, Helsinki, Finland
                Author information
                http://orcid.org/0000-0002-4368-4866
                Article
                621
                10.1186/s13058-015-0621-0
                4531540
                26243145
                53b20316-0ed1-4ca9-9e36-f3ee326cfea5
                © Sousa et al. 2015

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 24 April 2015
                : 21 July 2015
                Categories
                Research Article
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                © The Author(s) 2015

                Oncology & Radiotherapy
                Oncology & Radiotherapy

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