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

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.

Keywords: C-C chemokine receptor type 2; CC-chemokines ligand 2; CCL-2; CCR2; Cytokines; HCMV; IBC; IL-10; IL-13; IL-4; IL-8; INF-γ; Inflammatory breast cancer; Invasion; M1; M2; MCP-1; MMP-2; MMP-9; Macrophages; Motility; NF-κB; Nuclear factor kappa beta; PI3K; STAT3; TAMs; TNF-α; alternatively activated macrophages; c-Fms; classical activated macrophages; human cytomegalovirus; inflammatory breast cancer; interferon-γ; interleukin- 8; interleukin-10; interleukin-13; interleukin-4; macrophage colony stimulating factor-1 receptor; matrix metalloproteinases-2; matrix metalloproteinases-9; monocyte chemoattractant protein-1; phosphatidylinositol-3 kinas; signal transducer and activator of transcription; tumor associated macrophages; tumor necrosis factor-alpha.