Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties
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
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Both authors contributed equally to this work.