title: Human breast cancer cells educate macrophages toward the M2 activation status.
abstract: BACKGROUND
  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.
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, FI-70211, Kuopio, Finland. sofia.sousa@uef.fi.
authors.forename: Sofia
authors.initials: S
authors.lastname: Sousa
authors.affiliation: INSERM, UMR957, Equipe LIGUE 2012, Nantes, F-44035, France. regis.brion@univ-nantes.fr.|Université de Nantes, Nantes atlantique universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, F-44035, France. regis.brion@univ-nantes.fr.|CHU de Nantes, Nantes, F-44035, France. regis.brion@univ-nantes.fr.
authors.forename: Régis
authors.initials: R
authors.lastname: Brion
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland. minnamaija.lintunen@utu.fi.
authors.forename: Minnamaija
authors.initials: M
authors.lastname: Lintunen
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland. paukro@utu.fi.
authors.forename: Pauliina
authors.initials: P
authors.lastname: Kronqvist
authors.affiliation: Cell Imaging Core, Turku Centre for Biotechnology, University of Turku, and Åbo Akademi University, Turku, Finland. jouko.sandholm@btk.fi.
authors.forename: Jouko
authors.initials: J
authors.lastname: Sandholm
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, FI-70211, Kuopio, Finland. jukka.monkkonen@uef.fi.
authors.forename: Jukka
authors.initials: J
authors.lastname: Mönkkönen
authors.affiliation: Medical School, University of Tampere and Department of Oncology Tampere University Hospital, Tampere, Finland. pirkko-liisa.kellokumpu-lehtinen@uta.fi.
authors.forename: Pirkko-Liisa
authors.initials: PL
authors.lastname: Kellokumpu-Lehtinen
authors.affiliation: Laboratory of Molecular Oncology, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland. susanna.lauttia@helsinki.fi.
authors.forename: Susanna
authors.initials: S
authors.lastname: Lauttia
authors.affiliation: Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland. olli.tynninen@hus.fi.
authors.forename: Olli
authors.initials: O
authors.lastname: Tynninen
authors.affiliation: Laboratory of Molecular Oncology, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland. heikki.joensuu@hus.fi.|Comprehensive Cancer Center, Helsinki University Hospital, and Department of Oncology, University of Helsinki, Helsinki, Finland. heikki.joensuu@hus.fi.
authors.forename: Heikki
authors.initials: H
authors.lastname: Joensuu
authors.affiliation: INSERM, UMR957, Equipe LIGUE 2012, Nantes, F-44035, France. dominique.heymann@univ-nantes.fr.|Université de Nantes, Nantes atlantique universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, F-44035, France. dominique.heymann@univ-nantes.fr.|CHU de Nantes, Nantes, F-44035, France. dominique.heymann@univ-nantes.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Heymann
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, FI-70211, Kuopio, Finland. jorma.maatta@utu.fi.|Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Turku, Finland. jorma.maatta@utu.fi.
authors.forename: Jorma A
authors.initials: JA
authors.lastname: Määttä
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology; D016207:Cytokines / Q000378:metabolism; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000473:pathology*; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism
chemical_list: D016207:Cytokines; D016173:Macrophage Colony-Stimulating Factor; D020780:Matrix Metalloproteinase 9