Abstract
.BackgroundMacrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages.MethodsTHP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays.ResultsTHP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages.ConclusionOur results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible.
Highlights
Macrophages are one of the most important players in the tumor microenvironment
Extracellular vesicles (EVs) isolation and characterization EVs were isolated by sequential centrifugation, filtration and size exclusion chromatography (SEC) from the conditioned medium of the human isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured for 48 h
THP-1 monocytes and M0 macrophages take up SW480 and SW620-derived EVs First, the optimal amount of EVs for uptake experiments was determined by incubating THP-1 cells with Syto RNA Select labeled EVs at concentrations from 2 to 10 μg/mL for 1 h at 37 °C
Summary
Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. EVs contain several classes of biomolecules such as proteins, lipids and carbohydrates, as well as different kinds of RNA (mRNA, miRNA, piRNA, rRNA, tRNA fragments and other non-coding RNAs). Based on their cargo, EVs are regarded as molecular signal transfer mediators between cells, and are believed to be involved in the regulation of biological processes and to target cell functions and gene expression in recipient cells [2]. Cancer-derived EVs actively contribute to metastasis by modulating the microenvironment via degradation of the extracellular matrix and an increased production of pericellular hyaluronic acid, facilitating the invasion of cancer cells. The immunomodulatory effects of tumor derived EVs may reduce the activity of natural killer cells, cytotoxic T cells, and antigen presenting cells [8]
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