Background: 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. 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.
Methods: THP-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.
Results: THP-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.
Conclusion: Our 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.
Keywords: Extracellular vesicles; colorectal cancer; cytokines; endocytosis; macrophages; monocytes.