Faculty of Medicine, University of Latvia, Raina blvd. 19, Riga, LV-1568, Latvia.
Latvian Biomedical Research and Study Centre, Ratsupites iela 1, Riga, LV-1067, Latvia.
Cell Commun Signal. 2018 Apr 24;16(1):17. doi: 10.1186/s12964-018-0229-y.
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.
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.
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.
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.
巨噬细胞是肿瘤微环境中最重要的参与者之一。肿瘤相关巨噬细胞向促炎型 M1 或抗炎型 M2 的极化状态可能会影响癌症的进展和患者的生存。越来越多的证据表明，癌症来源的细胞外囊泡（EVs）被巨噬细胞摄取，并调节其表型和细胞因子谱。然而，癌症来源的 EVs 与不同分化和极化状态的单核细胞和巨噬细胞的相互作用还知之甚少。在本研究中，我们分析了原发性（SW480）和转移性（SW620）同源结直肠癌（CRC）细胞系衍生的 EVs 对单核细胞（M）、无活性巨噬细胞（M0）、M1 和 M2 极化巨噬细胞的摄取和功能影响。
用佛波醇-12-肉豆蔻酸-13-乙酸酯诱导 THP-1 单核细胞分化为 M0 巨噬细胞。然后，在 LPS、IFN-γ、IL-4 和 IL-13 的存在下，将 M0 巨噬细胞进一步极化成为 M1 和 M2 巨噬细胞。通过流式细胞术和荧光显微镜分析 SW480 和 SW620 衍生的 EV 的内化。通过流式细胞术、定量 PCR 和 Luminex 分析 EV 暴露后单核细胞和巨噬细胞免疫表型和分泌谱的变化。
THP-1 单核细胞和 M0 巨噬细胞有效地摄取了 SW480 和 SW620 衍生的 EVs，我们的结果表明，可能涉及网格蛋白依赖的内吞途径。有趣的是，SW480 和 SW620 衍生的 EVs 增加了 M0 巨噬细胞中 CD14 的表达，而 SW480 衍生的 EVs 降低了 M1 和 M2 极化巨噬细胞中 HLA-DR 的表达。此外，SW480 衍生的 EVs 显著增加了单核细胞和 M0 巨噬细胞中 CXCL10 的表达。相比之下，SW620 衍生的 EVs 诱导 M0 巨噬细胞中 IL-6、CXCL10、IL-23 和 IL-10 的分泌。然而，在巨噬细胞极化过程中，添加 CRC 细胞系衍生的 EVs 与 LPS、IFN-γ（M1）和 IL-4、IL-13（M2）刺激物一起，对 M1 和 M2 极化巨噬细胞中的细胞因子表达没有额外的影响。
我们的结果表明，CRC 细胞系衍生的 EVs 被摄取，并在单核细胞和无活性巨噬细胞中重新编程免疫表型和分泌谱，诱导混合的 M1 和 M2 细胞因子反应。尽管 CRC EVs 降低了 M1、M2 极化巨噬细胞中 HLA-DR 的表达，但对 M1 和 M2 极化巨噬细胞分泌谱的影响可以忽略不计。
