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. 2011 Sep 22:10:117.
doi: 10.1186/1476-4598-10-117.

Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells

Mei Yang  1 , Jingqi Chen, Fang Su, Bin Yu, Fengxi Su, Ling Lin, Yujie Liu, Jian-Dong Huang, Erwei Song
Affiliations

Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells

Mei Yang et al. Mol Cancer. .

Abstract

Background: Tumor-associated macrophages (TAMs) are alternatively activated cells induced by interleukin-4 (IL-4)-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs) circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells.

Results: We utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO) that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway.

Conclusions: We conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

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Figures

Figure 1
Figure 1
miR-223 was up-regulated in breast cancer cell lines co-cultured with IL-4-activated macrophages. (A) miRNA profiling in macrophages and the breast cancer cell line SKBR3 using the DiscovArray miRNA array. Three of the miRNAs that were present at high levels in macrophages but absent in SKBR3 cells are shown. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages). (B) Quantitative real-time PCR (qRT-PCR) confirmed a high expression level of hsa-miR-223 in macrophages but not in SKBR3 or MDA-MB-231 cells. * p < 0.05. (C) Co-culture of breast cancer cells with macrophages increased miR-223 levels in breast cancer cells. Breast cancer cell lines (SKBR3 and MDA-MB-231) were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages without direct cell-cell contact. miR-223 expression in SKBR3 and MDA-MB-231 cells was detected using qRT-PCR. Relative levels of miR-223 expression normalised to U6 rRNA levels are presented. * p < 0.05. (D) Functional assay of miR-223 in breast cancer cells. miR-223-targeting luciferase reporter containing a miR-223 complementary sequence within the 3'-UTR of the luciferase reporter gene was constructed. SKBR3 cells were transfected with the reporter gene and cultured alone (blank) or co-cultured with IL-4-activated macrophages. As controls, SKBR3 cells were co-transfected with the reporter gene and miR-NC, miR-660 or miR-223. Relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05.
Figure 2
Figure 2
Functional microRNA is shuttled from macrophages to breast cancer cells. (A) Schematic illustration of a miRNA transfer model. Macrophages were preloaded with Cy3 fluorescently-labeled miR-223 or the non-mammalian miRNA lin-4. A transwell system with a 0.4- μm pore size film, which allows small size materials (e.g., miRNAs), but not cells, to pass through, was used to separate SKBR3 cells from macrophages. (B) and (C) SKBR3 cells were cultured alone (blank) or co-cultured with macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. The Cy3 fluorescence signal in SKBR3 cells was determined by fluorescence microscopy (B), and the percentage of Cy3-positive cells in the images was calculated in (C). Arrowheads indicate Cy3-positive SKBR3 cells, and images are shown at 200×. (D) SKBR3 cells were cultured as shown in (B), and Cy3-positive cells (%) were quantified by flow cytometry. (E) A lin-4-targeting luciferase reporter gene carrying a lin-4 complementary sequence in the 3'-UTR was transfected into SKBR3 cells. SKBR3 cells were then co-cultured in a transwell with macrophages pre-transfected with miR-NC or lin-4. SKBR3 cells that were directly transfected with lin-4 were used as a control. Luciferase activities in SKBR3 cells were measured, and relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Figure 3
Figure 3
Exosomes secreted from macrophages shuttle miR-223 to breast cancer cells. (A) Exosomes secreted from unactivated and IL-4-activated macrophages were collected and observed with an electron microscope. Exosomes derived from unactivated macrophages (un-Mac-origin) were larger in size (100-250 nm) than those derived from IL-4-activated macrophages (IL-4-Mac-origin) (50-80 nm). Scale bars are as indicated. (B) The diameter of exosomes derived from unactivated and IL-4-activated macrophages were quantified. Data are averages of more than 10 exosomes from each group. (C) Levels of miR-223 expression in exosomes were quantified by qRT-PCR. ** p < 0.01. (D) and (E) Exosomes derived from IL-4-activated macrophages were more readily internalised by breast cancer cells. Exosomes derived from unactivated and IL-4-activated macrophages were labeled with CM-Dil and incubated with SKBR3 cells for the indicated times (0-40 h). Uptake of exosomes by cancer cells was visualized by confocal microscopy (representative images are shown in (E) and quantified by flow cytometry in (D)). (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Figure 4
Figure 4
Exosomal shuttling of miR-223 from macrophages to breast cancer cells promotes breast cancer cell invasion. (A) IL-4-activated macrophages promote breast cancer cell invasion. The breast cancer cell lines SKBR3 and MDA-MB-231 were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages. Approximately 24 to 48 h after co-culture, SKBR3 and MDA-MB-231 cells were subjected to an invasion assay. Data are averages of triplicates from more than three independent experiments and are presented as the number of invading cells per field. (B) miR-223 mimics enhanced the invasion of SKBR3 and MDA-MB-231 breast cancer cells. miR-223 was transfected into SKBR3 and MDA-MB-231 cells. Cell invasion was then determined by a transwell invasion assay. Relative invasion activities are presented as fold increases in the miR-223 group. The miR-NC group was normalized to 1.0. (C) Exposure to exosomes derived from macrophages resulted in similar effects on breast cancer invasion as the actual macrophages. SKBR3 cells were incubated with exosomes derived from unactivated or IL-4-activated macrophages and then treated with miR-NC ASO or miR-223 ASO. Cell invasion assays were performed as shown in (A). Data are averages of triplicates from three independent experiments and are presented as the number of invading cells per field. * p < 0.05; ** p < 0.01. (Un-Mac-exosome, exosomes originated from unactivated macrophages; IL-4-Mac-exosome, exosomes from IL-4-activated macrophages).
Figure 5
Figure 5
miR-223 targets the Mef2c-β-catenin pathway during breast cancer cell invasion. (A) miR-223 transfection inhibited Mef2c gene expression. HEK-293T cells were co-transfected with miR-223 and the Mef2c 3'-UTR-directed luciferase reporter (luciferase-Mef2c; Mef2c 3'-UTR cloned into the pMIR-REPORTER). Luciferase activities were measured as described in Figure 2. Cells transfected with miR-223 and luciferase vector, miR-NC and luciferase-Mef2c, or miR-223 and luciferase-miR-223C (miR-223 complementary sequence cloned in the 3'-UTR of luciferase reporter) were used as controls. Data are averages of triplicates from three independent experiments. * p < 0.05. (B) and (C) miR-223 reduced Mef2c expression and resulted in nuclear accumulation of β-catenin. SKBR3 cells were transfected with miR-223 or miR-NC (control). The expression of Mef2c and localisation of β-catenin in breast cancer cells were then determined by cellular fractionation followed by western blot analysis with anti-Mef2c, β-catenin and β-actin (B). The localisation of β-catenin in SKBR3 was visualized by indirect fluorescence microscopy. Briefly, cells were incubated with an anti-β-catenin antibody followed by an incubation with a FITC-conjugated anti-mouse secondary antibody. PI was used to visualise the nuclei (C). (scale bar: 20 μm).
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