Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties

https://doi.org/10.1016/j.biocel.2013.11.015Get rights and content

Abstract

Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.

Introduction

Inflammatory breast cancer (IBC) is the most aggressive and lethal form of breast cancer. Women often present with IBC at a young age (Nouh et al., 2011), are more likely to have metastatic disease at the time of diagnosis (Wedam et al., 2006) and have a shorter survival as compared to women with non-IBC (Chang et al., 1998). IBC is characterized by invasion into dermal lymphatic vessels where IBC cells form tumor emboli (Van Laere et al., 2006). Spreading of tumor emboli within lymphatic and blood vessels leads to distant metastasis and multi-organ failure in IBC patients (Tsoi et al., 2010). Dissemination of carcinoma cells can be regulated by cues from the inflammatory cells within the tumor microenvironment. Macrophages, which are the major inflammatory cells that infiltrate into breast tumors (Mukhtar et al., 2011, Pollard, 2008), contribute to high levels of growth factors, hormones, and cytokines (Aaltomaa et al., 1992, Georgiannos et al., 2003) and are designated as tumor associated macrophages (TAMs). Within the tumor microenvironment macrophages polarize into heterogeneous subpopulations depending on the type of external stimuli they receive (Cassetta et al., 2011). Among TAM subpopulations are: (1) ‘classical activated macrophages’ (M1), which are activated by pro-inflammatory agents such as interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) (Cassetta et al., 2011); (2) ‘alternatively activated macrophages’(M2) developed in response to IL-4 and IL-13 (Gordon and Martinez, 2010, Mantovani and Sica, 2010); and 3) ‘regulatory macrophages’ that express anti-inflammatory cytokines and increase tumor growth, invasion and metastasis (Mosser and Edwards, 2008). Classical activation induces the secretion of pro-inflammatory mediators by the macrophages and recruitment of T-cells as in an early inflammatory response (Ojalvo et al., 2009). M2 macrophages exhibit anti-parasite, immunosuppressive, wound healing and tissue remodeling properties (Gordon, 2003, Martinez et al., 2009). Indeed TAMs can regulate multiple mechanisms associated with dissemination of carcinoma cells. For instance, TAMs secrete proteolytic enzymes such as matrix metalloproteinases-2 and 9 (MMP-2 and MMP-9) that can degrade components of the basement membrane, thereby facilitating tumor cell intravasation and spreading in blood and lymphatic vessels (Hagemann et al., 2005, Mantovani et al., 2006). Increases in expression of MMPs and their inhibitors in TAMs have been found to correlate with distant metastasis of invasive ductal carcinomas (Gonzalez et al., 2007). The cysteine protease cathepsin B (Ibrahim et al., 2006) is expressed by TAMs in a transgenic mouse model for mammary carcinoma (Vasiljeva et al., 2006) and co-expressed with interleukin-10 (IL-10) in late stage lung cancer (Daurkin et al., 2011). We have previously shown that high levels of cathepsin B within the IBC microenvironment are associated with lymphatic metastasis (Nouh et al., 2011). Furthermore, TAMs secrete cytokines that control physiological mechanisms associated with tumor progression, i.e.,interleukin-8 (IL-8),which induces angiogenesis; tumor necrosis factor-alpha (TNF-α), which stimulates tumor growth and invasion (Dirkx et al., 2006), as well as immunosuppressive cytokines, i.e., monocyte chemoattractant protein-1 (MCP-1) or CC-chemokine ligand 2 (CCL-2) and IL-10(Daurkin et al., 2011). In fact TAMs play crucial roles in the dissemination of breast cancer cells. This is evident from intravital imaging which has provided new insights into how subpopulations of TAMs patrol inside blood vessels in the tumor microenvironment and at the tumor margins (Egeblad et al., 2008). Thus macrophages are considered to be potential diagnostic and therapeutic targets (Mukhtar et al., 2011). Therapeutic strategies include targeting macrophage recruitment to the tumor site by CCL-2 neutralizing antibodies (Balkwill and Mantovani, 2010); or altering macrophage development by targeting macrophage colony stimulating factor-1 receptor (c-Fms) using the tyrosine kinase inhibitor imatinib (Dewar et al., 2005).

Although macrophages have been identified as major cellular components of the non-IBC microenvironment their role in IBC is not well understood (Kleer et al., 2000). Herein, we show that IBC is characterized by high infiltration and venous drainage of macrophages that secrete cytokines different from those secreted by macrophages from non-IBC patients. Moreover, we identified major cytokines that may contribute to IBC invasion and motility and can be therapeutically targeted.

Section snippets

Patients

For the purpose of patient enrollment in this study, we obtained Institutional Review Board (IRB) approval from the ethics committee of Ain-Shams University, Cairo, Egypt and the National Cancer Institute (NCI), Cairo University, Giza, Egypt. Patients were enrolled from outpatient breast clinics of Ain Shams University hospitals and NCI Cairo University during the period of January 2010–January 2012. All patients signed consent form including approval for publication of the study results before

Patient clinical and pathological characteristics

Clinical and pathological characterization of IBC and non-IBC patients is shown in Table 1. Women with IBC were more likely to present with 4 or more positive lymph nodes than women with non-IBC. All IBC patients showed positive tumor emboli in comparison to 11% of non-IBC patients.

Inflammatory breast carcinoma tissues characterized by high infiltration of CD14+ cells

We evaluated the level of infiltration of monocytes/macrophages in non-IBC and IBC paraffin embedded breast carcinoma tissues using monoclonal antibodies specific for CD14+ (monocyte differentiation marker) and CD68+

Discussion

The tumor microenvironment is characterized by infiltration of monocytes/macrophages that play distinct roles in tumor progression and metastasis (Cassetta et al., 2011). The notion that macrophages are “obligate partners for tumor cell migration, invasion, and metastasis” (Condeelis and Pollard, 2006) and that TAMs induce an invasive phenotype (Mantovani et al., 2006, Sica et al., 2006) is evident from several studies. Macrophages have been found to attract carcinoma cells to intravasate into

Conflict of interest

The authors declare that they have no competing interests.

Author's contribution

EAG, MES, MAN and MMM carried out the experiments. MMM, BFS, RJS, MAN and MES made substantial contributions to concept and design of experiments as well as to the drafting and/or revising of the manuscript. All authors have read and approved the manuscript.

Acknowledgements

This work was conducted at Cancer Biology Research Laboratory (CBRL), Department of Zoology, Faculty of Science, Cairo University, Egypt. We acknowledge the contribution of Dr. Sayed F. Abdelwhab and Dr. Maha Sobhy, the Egyptian Company for Blood Transfusion Services (Egyblood)-VACSERA, Giza, Egypt for their help in some flow cytometric experiments. We thank Ms. Amal Youns, postgraduate student at CBRL, for her help in the preparation of clinical-pathological data. We also thank Ms. Haba

References (67)

  • L.S. Ojalvo et al.

    High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors

    Am J Pathol

    (2009)
  • A. Sica et al.

    Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy

    Eur J Cancer

    (2006)
  • G. Soria et al.

    The inflammatory chemokines CCL2 and CCL5 in breast cancer

    Cancer Lett

    (2008)
  • P. Szlosarek et al.

    Tumour necrosis factor-alpha as a tumour promoter

    Eur J Cancer

    (2006)
  • A. Valster et al.

    Cell migration and invasion assays

    Methods

    (2005)
  • D. Al-Raawi et al.

    Membrane type-1 matrix metalloproteinase (MT1-MMP) correlates with the expression and activation of matrix metalloproteinase-2 (MMP-2) in inflammatory breast cancer

    Int J Clin Exp Med

    (2011)
  • A. Astanehe et al.

    The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1

    Oncogene

    (2009)
  • F. Balkwill et al.

    Cancer and inflammation: implications for pharmacology and therapeutics

    Clin Pharmacol Ther

    (2010)
  • P. Bonnier et al.

    Inflammatory carcinomas of the breast: a clinical, pathological, or a clinical and pathological definition?

    Int J Cancer

    (1995)
  • I. Caras et al.

    Influence of tumor cell culture supernatants on macrophage functional polarization: in vitro models of macrophage-tumor environment interaction

    Tumori

    (2011)
  • L. Cassetta et al.

    Macrophage polarization in health and disease

    Sci World J

    (2011)
  • S. Chang et al.

    Inflammatory breast carcinoma incidence and survival: the surveillance, epidemiology, and end results program of the National Cancer Institute, 1975–1992

    Cancer

    (1998)
  • I. Daurkin et al.

    Tumor-associated macrophages mediate immunosuppression in the renal cancer microenvironment by activating the 15-lipoxygenase-2 pathway

    Cancer Res

    (2011)
  • S. Dawood et al.

    International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment

    Ann Oncol

    (2010)
  • D.G. DeNardo et al.

    Immune cells as mediators of solid tumor metastasis

    Cancer Metastasis Rev

    (2008)
  • A.E. Dirkx et al.

    Monocyte/macrophage infiltration in tumors: modulators of angiogenesis

    J Leukoc Biol

    (2006)
  • M. Egeblad et al.

    Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

    Dis Model Mech

    (2008)
  • M. El-Shinawi et al.

    Capturing and characterizing immune cells from breast tumor microenvironment: an innovative surgical approach

    Ann Surg Oncol

    (2010)
  • M. El-Shinawi et al.

    Human cytomegalovirus infection enhances NF-kappaB/p65 signaling in inflammatory breast cancer patients

    PLoS ONE

    (2013)
  • F. Forozan et al.

    Molecular cytogenetic analysis of 11 new breast cancer cell lines

    Br J Cancer

    (1999)
  • C. Genestie et al.

    Comparison of the prognostic value of Scarff–Bloom–Richardson and Nottingham histological grades in a series of 825 cases of breast cancer: major importance of the mitotic count as a component of both grading systems

    Anticancer Res

    (1998)
  • S.H. Giordano et al.

    Inflammatory breast cancer: clinical progress and the main problems that must be addressed

    Breast Cancer Res

    (2003)
  • L.O. Gonzalez et al.

    Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse

    Br J Cancer

    (2007)
  • Cited by (81)

    • The immunogram of inflammatory breast cancer

      2023, Cancer Treatment Reviews
    • IL-8 secreted by tumor associated macrophages contribute to lapatinib resistance in HER2-positive locally advanced breast cancer via activation of Src/STAT3/ERK1/2-mediated EGFR signaling

      2021, Biochimica et Biophysica Acta - Molecular Cell Research
      Citation Excerpt :

      Previously we showed that monocytes contribute to the aggressive behavior of inflammatory breast cancer (IBC), we showed that human monocyte secrete IL-8 that induce expression of mesenchymal marker fibronectin; a mechanism modulated by PI3k/AkT signaling pathway [41]. In addition, we also demonstrated that TAM drained from breast TME through axillary tributaries secrete cytokines, IL-8 is one of the most prominent secreted cytokines, that induce motility and invasion of IBC cells in-vitro [43]. Considering all of the above results introduced by different studies and our previous studies herein we assumed that resistance to lapatinib treatment in LABC patients may be due to cytokines secreted by TAMs that activate RTKs and associated signaling molecules used to be inhibited by lapatinib treatment.

    • Induction of heparanase via IL-10 correlates with a high infiltration of CD163+ M2-type tumor-associated macrophages in inflammatory breast carcinomas

      2020, Matrix Biology Plus
      Citation Excerpt :

      About 10–15 ml blood was collected from tumor microenvironment of IBC or non-IBC patients, through axillary tributaries during surgery in heparinized tubes. Collected blood was transferred directly to the laboratory for isolation of tumor-associated macrophages (TAMs) using density-gradient centrifugation as we described before [30]. Briefly, mononuclear cells were suspended in RPMI 1640 medium containing 1% heat-inactivated FBS at density of 1 × 106 cells/ml.

    View all citing articles on Scopus
    1

    Both authors contributed equally to this work.

    View full text