Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Abstract

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

Figures

Figure 1. Analysis of protein expression in circulating exosomes from melanoma subjects

(a) Representative electron microscopic image of exosomes derived from the plasma of a melanoma subject. Scale bar, 100 nm. (b) Kaplan-Meier survival curve showing cumulative probabilities in Stage IV subjects over 42 months of follow-up according to total protein (micrograms) in isolated circulating exosomes per milliliter of plasma analyzed (n = 15). P values were calculated using a log-rank test. (c) Representative Western blot of TYRP2, VLA-4, HSP70, HSP90 and HSC70 proteins in circulating exosomes isolated from the plasma of melanoma subjects (Stages I, III and IV) and healthy controls. Arrow indicates a specific HSP90 isoform found in 70% of melanoma subjects. GAPDH was used as a loading control. (d) Statistical analysis of western blot densitometry of signature proteins in circulating exosomes relative to GAPDH. Controls (n = 9); stage I (n = 2); stage III (n = 7); stage IV (n = 18). P values were calculated by ANOVA. (e) Statistical analysis of western blot densitometry for TYRP2 expression in circulating exosomes relative to GAPDH in a retrospective series of frozen plasma derived from stage III melanoma subjects (n = 29) who had been followed for 4 years to evaluate disease progression (NED = no evidence of disease, POD = progression of disease), (n = 29); P < 0.001 using Mann-Whitney U test.

Figure 2. Role of tumor-derived exosomes in metastasis

(a) Measurement of the total protein per million cells in exosomes isolated from human and mouse melanoma cells in culture. Error bars represent s.e.m. (b) Confocal microscopic analysis of B16-F10 exosome tissue distribution (green) 5 min (lung, left panel) or 24 h (lung and BM, right panels) after tail vein injection. Scale bar, 50 μm. (c) Analysis of lung endothelial permeability following fluorescently-labeled dextran perfusion (red) 24 h after tail vein injection of B16-F10 exosomes, conditioned medium or control particles. Scale bar, 50 μm. (d) Analysis of primary tumor growth (left panel) after subcutaneous flank injection of B16-F10mCherry cells in WT mice treated with B16-F10 exosomes for 3 weeks (n = 6 mice per group; error bars represent s.e.m.; * P < 0.05 by ANOVA). Red arrows indicate exosome injections. The black arrow denotes the timepoint (day 19) at which lung micrometastatic lesions were analyzed. Lung micrometastases (mCherry+, middle panels, scale bar, 50 μm) were quantified by immunofluorescence (right panel). (e) Analysis of primary tumor growth after subcutaneous flank injection of B16-F10-luciferase cells in mice pre-treated with 5μg of B16-F10 and B16-F1 exosomes three times a week for 28 d. (f) Metastatic burden was quantified by luciferin photon flux at 21 d post-tumor injection (left panel). Scale bar, 200 μm. Quantification of total photon flux in lungs and bones (right panel), n = 10 mice per group; error bars represent s.e.m.; *P < 0.05 by ANOVA.

Figure 3. Role of tumor-derived exosomes in BM cell education and metastasis

(a) Schematic of the experiment performed to analyze the influence of tumor exosomes on BM cell education and metastasis (GFP+= green fluorescent protein). (b) Analysis of primary tumor growth after subcutaneous flank injection of B16-F10mCherry cells in mice transplanted with B16-F10 exosome-educated BM (BM-educated). BM derived from mice treated with control particles (BM-control) was used in parallel, n = 5 mice per group; error bars represent s.e.m.; ***P < 0.001 by ANOVA. (c) Confocal microscopic analysis of BMDCs (GFP+) and vasculature (lectin-red) in primary tumors from BM-educated mice and controls (top panels). Scale bar, 200 μm. Quantification of vasculature and total BMDCs is shown below (lower panels), n = 5 mice per group; error bars represent s.e.m. P value by ANOVA. (d) BMDCs (GFP+) and tumor B16-F10 cells (mCherry+-red) in lung metastatic lesions at 28 d post-tumor injection (top panels). Scale bar, 50 μm. Quantification of metastatic area, tumor burden and total BMDCs is shown in the lower panels (left), n = 5 mice per group; error bars represent s.e.m. P value by ANOVA. Macroscopic analysis of lung metastases at day 35 is shown in the right panel. Scale bar, 200 mm. (e) Flow cytometric analysis of indicated BM progenitor cell populations in mice educated with B16-F10 and B16-F1 exosomes or control particles for 28 d (n = 5 mice per group; error bars represent s.e.m.; NS = not significant by ANOVA).

Figure 4. Met analysis in tumor and BM cells

(a) Western blot analysis of Met and phospho-Met in B16-F1 and B16-F10 exosomes and cells (b) QRT-PCR analysis of Met and Cd44 in lineage-negative BM cells after B16-F10 or B16-F1 exosome education; error bars represent s.e.m.; NS = not significant by ANOVA. (c) Flow cytometric analysis of c-Kit and Met expression on BM cells after overnight incubation with fluorescently labeled exosomes (20 μg ml−1, PKH26+). FL2 fluorescence indicates exosome uptake (right panel). (d) Flow cytometric analysis of Met expression in BM c-Kit+Tie2+ cells (upper panel) and Lin–c-Kit+Tie2+ circulating blood cells (lower panel) of mice educated with B16-F10 and B16-F1 exosomes (red area=percentage of Met+ cells). Error bars represent s.e.m.; NS = not significant by ANOVA. (e) Analysis of metastasis in mice educated with 5μg of B16-F10, B16-F10shMet and control. Scale bar, 200 μm. Quantification of total photon flux 21d post B16-F10-luciferase cell injection (lower panel). Error bars represent s.e.m.; P value by ANOVA. (f) Multiplex protein analysis of MET and phospho-MET (Tyr1349) in the circulating exosomes from a retrospective series of frozen plasma derived from melanoma subjects and controls. Controls (n = 7); Stage III (n = 24); Stage IV (n = 15). P values by ANOVA. (g) Flow cytometric analysis depicting the percentage of MET+ BM progenitor cells in the blood of individuals with melanoma. Controls (n = 7); stage I–III (n = 10); stage IV (n = 9). Error bars represent s.e.m; P values by ANOVA.

Figure 5. Disrupting Rab27a expression reduces exosome release, tumor growth, and metastasis

(a) QRT-PCR analysis of RAB genes in melanoma (SK-Mel-#), breast cancer (MCF7, MDA-MB-231, SkBr3) and pancreatic adenocarcinoma (AsPc1) human cell lines. Red denotes high (≥ 2—fold), white intermediate (< 2—fold and > 1.5—fold), and blue low (≤ 1.5—fold) RAB expression in melanoma relative to breast cancer and pancreatic cell lines. (b) Measurement of the total protein in the exosomes secreted per million human melanoma cells in culture. (c) QRT-PCR analysis of Rab27a expression after shRNA knockdown of Rab27a in B16-F10 and SK-Mel-28 cell lines. (d) Measurement of exosome protein per million cells after shRNA knockdown of Rab27a in B16-F10 and SK-Mel-28 cell lines. Control scramble shRNA and parental cells were used as a reference. (e) Characterization and densitometric analysis of conditioned medium derived from B16-F10-shScramble and -shRab27a cell lines. (f) Analysis of primary tumor growth and metastasis in shScramble, sh-Rab27a- B16-F10 and SK-Mel28 cell lines subcutaneously injected into the flank of C57BL/6 and NOD SCID mice, respectively. Metastases were macroscopically counted (B16-F10) or quantified by QRT-PCR for mCherry (SK-Mel-28), n = 5 mice per group; error bars represent s.e.m.; ***P < 0.001 by ANOVA. (g) Analysis of BMDCs (GFP+-green) and tumor cells (mCherry-red) in B16-F10-shScramble and -shRab27a primary tumors (upper panel, scale bar, 200 μm) and lungs (lower panels, scale bar, 50 μm). Quantification of the metastatic area and total BMDCs is shown on the right, n = 5 mice per group; Error bars represent s.e.m; P value by ANOVA.