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. 2017 Dec 13;9(7):7398-7410.
doi: 10.18632/oncotarget.23238. eCollection 2018 Jan 26.

Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis

Yin Ji Piao  1   2 , Hoe Suk Kim  1 , Eun Hye Hwang  1 , Jisu Woo  1 , Meihua Zhang  1   3 , Woo Kyung Moon  1   2
Affiliations

Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis

Yin Ji Piao et al. Oncotarget. .

Abstract

Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro. In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC.

Keywords: exosome; lymph node; macrophage; metastasis; triple-negative breast cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing interests.

Figures

Figure 1
Figure 1. Generation of stable MDA-MB-231 cells overexpressing the exosomal CD63-RFP fusion protein and analysis of purified RFP-tagged exosomes
(A) Confocal images of CD63-RFP–transduced MDA-MB-231 cells. (B) Confocal image of purified RFP-tagged exosomes. (C) NanoSight analysis of the size and concentration of purified RFP-tagged exosomes. (D) Western blot of CD63, ALIX, calnexin, and RFP in the purified RFP-tagged exosome (EXO) and the lysates of MDA-MB-231/CD63-RFP cells (CL).
Figure 2
Figure 2. TNBC cell migration and proliferation is enhanced by TNBC cell–derived exosomes
(A) Confocal images of transportation of RFP-tagged exosomes in direct co-culture with MDA-MB-231/CD63-RFP cells and MDA-MB-231/GFP cells for 24 hours. (B) Confocal image of RFP-exosomes (EXO) taken up by MDA-MB-231/GFP cells after administration of RFP-tagged exosomes (10 µg/mL) for 24 hours. (C)Wound-healing assay in MDA-MB-231 cells treated with RFP-tagged exosomes (5–10 µg/mL) or PBS for 15 to 21 hours. (D) Proliferation assay of MDA-MB-231 cells treated with RFP-tagged exosomes (10–50 µg/mL) or PBS for 24 to 48 hours.
Figure 3
Figure 3. Induction of M1/M2 polarization by TNBC cell–derived exosomes in vitro and in vivo
(A) Confocal images of RFP-tagged exosome transportation in direct co-culture with MDA-MB-231/CD63-RFP cells and RAW264.7/GFP cells for 24 hours. (B) Proliferation assay in RAW264.7 cells treated with RFP-tagged exosomes (30 or 50 µg/mL) or PBS for 24 to 48 hours. (C) Trans-well migration assay in RAW264.7 cells treated with RFP-tagged exosomes (EXO, 5–10 µg/mL) or PBS for 24 to 48 hours. (D) Immunostaining of CD206 and NOS2 in RAW264.7 cells cultivated with MDA-MB-231/CD63-RFP cells in the trans-well system for 24 hours. (E and F) Western blot and real-time RT-PCR of arginase-1, CD206, and NOS2 in RAW264.7 cells administered RFP-tagged exosomes (10 µg/mL) or PBS for 24 to 48 hours. (G) Immunostaining images of CD63, CD206, and NOS2 in axillary LNs removed from mice at 3 hours after intravenous injection of RFP-tagged exosomes (100 µg) or PBS. (H) Quantitative immunostained area (mean ± S.E.) of CD63, CD206, and NOS2. ND indicates no detection.
Figure 4
Figure 4. Noninvasive BLI and US-guided PAI of primary tumor growth and axillary LN metastasis promoted by cancer cell–derived exosomes in TNBC models
(A) A flowchart depicting the experimental design in an orthotropic tumor model. (B) Representative BLI of primary tumors of mice intravenously injected with PBS or RFP-tagged exosomes (EXO, 10 µg, 10 injections at 2 day-intervals) at 2, 4, and 6 weeks after fat pad injection with MDA-MB-231/Luc-GFP cells. (C) Total photon flux (mean ± S.E.) measured from primary tumors. (D and F) Representative US-guided PAI of primary tumor and axillary LNs of mice intravenously injected with PBS or RFP-tagged exosomes before and 4 hours and 24 hours after intratumor injection of anti–EGFR-GN (7.7 mg/kg). (E and G) PA signals (mean ± S.E.) measured from primary tumors and axillary LNs. (H and I) Representative ex vivo US-guided PAI and GFP confocal images of axillary LNs isolated from mice intravenously injected with PBS or RFP-tagged exosomes.
Figure 5
Figure 5. Histological analysis of axillary LN metastasis promoted by cancer cell–derived exosomes in TNBC models
After follow-up of tumor growth and axillary LN metastasis by use of biweekly BLI and US-guided PAI by intratumor injection with anti–EGFR-GNs (7.7 mg/kg), axillary LNs were isolated from tumor-bearing mice injected with RFP-tagged exosomes (EXO) or PBS at 6 weeks. (A) H&E and silver staining images for the investigation of anti–EGFR-GNs accumulation in axillary LNs. (B). Immunostaining images of EGFR, CK18/8/19, and GFP for the evaluation of metastasis in axillary LNs. (C) Immunostaining images of CD206 and NOS2 for the evaluation of macrophage M2/M1 polarization in axillary LNs. Immunostaining images of CD63 for the analysis of cancer-derived exosome distribution in axillary LNs. (D) Quantitative immunostained area (mean ± S.E.) of CD63, CD206, and NOS2.
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