Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-κB

Abstract

Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression.

Figure 1. Macrophages activate NF-κB in response to breast cancer-derived exosomes.

(a) Representative EM images of exosomes derived from MCF10A, MDA-MB-231 (abbreviated to MDA-231 or 231 in Figures), and MCF7 cells. Inset in MCF10A panel shows enhanced detail of cup-like morphology. Scale bar: 200 nm. (b) Western blot of indicated exosomes (exo) for CD63. Membranes/blots are cropped by molecular weights to show the proteins of interest. Samples from the three cell lines were run separately as the protein abundance was not compared to each other. The gels were run under the same experimental conditions. The original whole blot pictures are available in Supplementary Fig. S3a. (c) Raw 264.7 cells incubated with DiI-labeled exosomes derived from indicated cell types were analyzed by flow cytometry. Accumulation of DiI fluorescence, indicated along the X-axis with DiI⁺ percentage noted, reflects association of exosomes with Raw macrophages. (d) Luciferase fold induction of RawkB cells fed exosomes from indicated cells types, reflecting NF-κB activation. *p < 0.001. (e) Western blot of RawkB cells incubated with LPS or with exosomes from cell types as specified. Treatments using exosomes were carried out for 20 min after which cells were lysed and Western blotted using indicated antibodies reflecting NF-κB activation status. The fold difference in band intensities was quantified and indicated under the image. Membranes/blots are cropped by molecular weights to show the proteins of interest. The gels were run under the same experimental conditions. The original whole blot pictures are available in Supplementary Fig. S3b.

Figure 2. Macrophages secrete inflammatory cytokines in response to breast-cancer derived exosomes in vitro.

(a) Cytokine array identifies IL-6, CCL2, TNFα, and GCSF as the inflammatory cytokines whose expression is most highly induced following exposure to MDA-231 and MCF7 exosomes, but not MCF10A exosomes. The original full-length blots are shown. (b–d) Quantitative RT-PCR for IL-6 and TNFα in (b) RawkB cells, (c) THP-1 cells, and (d) bone marrow derived macrophages following administration of exosomes derived from MDA-231 (231) and MCF10A (10A) cells. (e) Quantitative RT-PCR for IL-6 and TNFα in RawkB cells pre-treated with 40 μM Bay 11-7082 for 1 h followed by stimulation with 231 exosomes for 6 h. *p < 0.001.

Figure 3. Macrophages secrete inflammatory cytokines in response to breast-cancer derived exosomes in vivo.

(a–b) DiI-labeled MDA-231 exosomes administered intravenously associate with lung macrophages and induce inflammatory cytokine production. Following tail-vein injection of DiI-labeled exosomes, lung tissue was harvested and dissociated. (a) Flow cytometric analysis following staining with anti-F4/80 antibody revealed a population of lung macrophages associating with MDA-231 exosomes (top right quadrant). (b) Quantitative RT-PCR for TNFα was carried out using RNA isolated from F4/80⁺ cells sorted on DiI^+/− fluorescence. *p < 0.001. (c) Representative immunofluorescence imaging of brain tissue from mice administered DiI-labeled MDA-231 exosomes. Left color-merged image shows F4/80⁺ macrophages in green, DAPI in blue, and DiI exosomes in red. Scale bar: 5 μm. Insets show a higher magnification. Right color-merged image shows IL-6 in green, DAPI in blue, and DiI exosomes in red. Grayscale of each color channel is shown in bottom panels. (d) Representative immunofluorescence imaging of axillary lymph node tissue from mice with mammary fat pad xenograft MDA-231 primary tumor. White box highlights CD68⁺ macrophage with associated human CD63⁺ exosomes, which is expressing IL-6 (green, right panel). Grey box highlights CD68⁺ macrophage without associated exosomes and not expressing IL-6. Scale bar: 10 μm.

Figure 4. Stimulation of macrophages is mediated by proteins on the surface of cancer-derived exosomes.

(a) RawkB cells were fed MDA-231 (231) or MCF10A (10A) exosomes for indicated durations followed by assessment of luciferase activity. (b) 100 μg/mL of proteinase K (ProtK) was used to treat intact or sonicated exosomes for 1 h at 37°C. Inactivation of ProtK was carried out by treatment with 5 mM PMSF. Exosome samples were used to treat RawkB cells for 4 h followed by assessment of luciferase activity. (c) Exosomes were incubated with 50 U of DTT-activated SLO on ice, washed with PBS, and used to treat RawkB cells for 4 h followed by assessment of luciferase activity. (d) Exosomes were treated with 100 mM glycine, pH 2.5, neutralized with 2 M Tris, pH 8.0, and used to treat RawkB cells for 4 h followed by assessment of luciferase activity. (e) IL-6 expression levels were determined by quantitative RT-PCR in RawkB cells treated with exosomes as indicated. *p < 0.001.

Figure 5. Stimulation by cancer-derived exosomes occurs through TLR2 on macrophages.

MDA-231 (231) or MCF10A (10A) exosomes were fed to bone marrow-derived macrophages generated from indicated mouse strains. (a) IL-6 and (b) TNFα expression levels were determined by quantitative RT-PCR. *p < 0.001. (c) Endosomally localized TLRs in RawkB cells were inhibited by a 2-h pretreatment with indicated concentrations of Chloroquine. Stimulation was subsequently carried out by feeding cells with exosomes or TLR ligands for 4 h followed by assessment of luciferase activity. *p < 0.001 compared to the PBS (control) group. (d) RawkB cells were treated with MDA-231 exosomes or PBS in the presence of a TLR2 neutralizing antibody or a control antibody at a final concentration of 30 ng/ml. Luciferase activity was assessed and compared to the PBS group. *p < 0.001.

Figure 6. Palmitoylated proteins on the surface of cancer-derived exosomes contribute to stimulation of macrophages.

(a) Exosomes isolated from MDA-231 cells were treated with 1% H₂O₂ for 4 h at room temperature to oxidize lipoprotein thioesters to sulfoxide and subsequently washed with PBS to eliminate residual H₂O₂. Treated and non-treated exosomes were administered to RawkB cells for 4 h followed by assessment of luciferase activity. (b) Exosomes isolated from MDA-231 cells were treated with 1 M hydroxylamine (HA) for 1 h at room temperature to remove S-acyl groups and subsequently washed with PBS to eliminate residual HA. HA-treated and vehicle-treated exosomes were administered to RawkB cells for 4 h followed by assessment of luciferase activity. (c) Exosomes isolated from MDA-231 cells treated with 2-bromopalmitate (2BP) were administered to RawkB cells for 4 h followed by assessment of luciferase activity. (d) Identification of palmitoylated membrane proteins in MDA-231 and MCF10A cells was carried out by isolating the membrane fraction from 5 × 10⁷ cells and performing an acyl-biotin exchange using HPDP-biotin. Streptavidin-conjugated magnetic beads were used to enrich the biotinylated (previously palmitoylated) proteins. Washed beads were reduced and alkylated, allowing for detachment of proteins from the beads, followed by trypsin digestion and HPLC-MS. *p < 0.001.