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

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.

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. 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. 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. 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. 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.