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

Journal name:
Nature Medicine
Volume:
18,
Pages:
883–891
Year published:
DOI:
doi:10.1038/nm.2753
Received
Accepted
Published online
Corrected online

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 CD45C-KITlow/+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.

At a glance

Figures

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

    (a) A representative electron microscopic image of exosomes derived from the plasma of a subject with melanoma. Scale bar, 100 nm. (b) Kaplan-Meier survival curves showing the cumulative survival probabilities in subjects with stage 4 melanoma over 42 months of follow up according to the total amount of protein (micrograms) in isolated circulating exosomes per milliliter of plasma analyzed (n = 15). Overall P values were calculated using a log-rank test. (c) Representative western blot of TYRP2, HSP70, VLA-4, HSP90 and heat shock cognate protein 70 (HSC70) in circulating exosomes isolated from the plasma of subjects with melanoma (at stage 1, 3 or 4, as indicated) and healthy controls. The arrow indicates a specific HSP90 isoform that was found in 70% of the stage 4 subjects with melanoma. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (d) Statistical analysis of the western blot densitometry of signature proteins in circulating exosomes relative to GAPDH. Controls, n = 9; stage 1, n = 2; stage 3, n = 7; stage 4, n = 18. P values were calculated by analysis of variance (ANOVA). (e) Statistical analysis of the western blot densitometry for TYRP2 expression in circulating exosomes relative to GAPDH in a retrospective series of frozen plasma derived from subjects with stage 3 melanoma (n = 29) who had been followed for 4 years to evaluate disease progression (NED, no evidence of disease; POD, progression of disease). P < 0.001 using Mann-Whitney U test.

  2. Role of tumor-derived exosomes in metastasis.
    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, s.e.m. (b) Confocal microscopic analysis of B16-F10 exosome tissue distribution (green) 5 min (lung, left) or 24 h (lung and bone marrow, right) after tail vein injection. Scale bars, 50 μm. (c) Analysis of lung endothelial permeability after perfusion with fluorescently labeled dextran (red) 24 h after tail vein injection of B16-F10 exosomes (exo), conditioned medium or control particles. Scale bar, 50 μm. (d) Analysis of primary tumor growth (left) after subcutaneous flank injection of B16-F10mCherry cells in wild-type mice treated with 10 μg of B16-F10 exosomes for 3 weeks. n = 6 mice per group. Error bars, s.e.m. *P < 0.05 by ANOVA. Red arrows indicate exosome injections. The black arrow denotes the time point (day 19) at which lung micrometastatic lesions were analyzed. Lung micrometastases (mCherry+, middle, scale bars, 50 μm) quantified by immunofluorescence (right). (e) Analysis of primary tumor growth after subcutaneous flank injection of B16-F10–luciferase cells in mice pretreated with 5 μg of B16-F10 and B16-F1 exosomes as described in the text. Error bars, s.e.m. (f) Metastatic burden quantified by luciferin photon flux at 21 d after tumor injection (left). Scale bars, 200 μm. Quantification of total photon flux in lungs and bones (right). n = 10 mice per group. Error bars, s.e.m. *P < 0.05 by ANOVA.

  3. Role of tumor-derived exosomes in bone marrow cell education and metastasis.
    Figure 3: Role of tumor-derived exosomes in bone marrow cell education and metastasis.

    (a) Schematic of the experiment performed to analyze the influence of tumor exosomes on bone marrow (BM) cell education and metastasis. (b) Analysis of primary tumor growth after subcutaneous flank injection of B16-F10mCherry cells in mice transplanted with B16-F10 exosome-educated bone marrow (BM - educated). Bone marrow derived from mice treated with control particles (BM - control) was used in parallel. n = 5 mice per group. Error bars, s.e.m. ***P < 0.001 by ANOVA. (c) Confocal microscopic analysis of BMDCs (GFP+) and vasculature (lectin, red) in primary tumors from bone marrow–educated mice and controls (top). Scale bars, 200 μm. A quantification of the vasculature and total number of BMDCs is shown below. n = 5 mice per group. Error bars, s.e.m. The P values were determined by ANOVA. (d) BMDCs (GFP+) and tumor B16-F10 cells (mCherry+, red) in lung metastatic lesions from bone marrow–educated mice and controls at 28 d after tumor injection (top). Scale bars, 50 μm. A quantification of the metastatic area, tumor burden and total number of BMDCs is shown on the bottom left. n = 5 mice per group. Error bars, s.e.m. P values were determined by ANOVA. On the bottom right is a macroscopic analysis of lung metastases at day 35. Scale bars, 200 μm. (e) Flow cytometric analysis of the indicated bone marrow progenitor cell populations in mice educated with 10 μg B16-F10 and B16-F1 exosomes or control particles three times a week for 28 d. n = 5 mice per group. Error bars, s.e.m. NS, not significant by ANOVA.

  4. MET analysis in tumor and bone marrow cells.
    Figure 4: MET analysis in tumor and bone marrow cells.

    (a) Western blot analysis of Met and pMet in B16-F1 and B16-F10 exosomes and cells. pMet (Tyr1234/1235), Met phosphorylated at Tyr1234 and Tyr1235. (b) Quantitative real time PCR analysis of Met and Cd44 in lineage-negative bone marrow cells after B16-F10 or B16-F1 exosome injection. Error bars, s.e.m. NS, not significant by ANOVA. Lin, lineage negative (see Online Methods); F10, B16-F10; F1, B16-F1. (c) Flow cytometric analysis of c-Kit and Met expression on bone marrow cells after overnight incubation with fluorescently labeled exosomes with red fluorescent cell linker (PKH26+). Fluorescence in the red channel (FL2) indicates exosome uptake (right). (d) Flow cytometric analysis of Met expression in c-Kit+Tie2+ bone marrow cells (top) and Linc-Kit+Tie2+ circulating blood cells (bottom) of mice injected with 10 μg of B16-F10 and B16-F1 exosomes three times a week over 28 d (the red area indicates the percentage of Met+ cells). Error bars, s.e.m. NS, not significant by ANOVA. (e) Analysis of metastasis after subcutaneous flank injection of B16-F10–luciferase cells in mice pretreated with 5 μg of B16-F10 or B16-F10shMet exosomes or control particles three times a week over 28 d. Scale bars, 200 μm. Metastatic burden quantified by luciferin photon flux at 21 d after tumor injection (bottom). Error bars, s.e.m. P values were determined by ANOVA. (f) Multiplex protein analysis of MET and pMET (phosphorylated at Tyr1349) in the circulating exosomes isolated from a retrospective series of frozen plasma derived from subjects with melanoma and controls. Controls, n = 7; stage 3, n = 24; stage 4, n = 15. P values were determined by ANOVA. (g) Flow cytometric analysis depicting the percentage of MET+ bone marrow progenitor cells in the blood of individuals with melanoma. Controls, n = 7; stage 1–3, n = 10; stage 4, n = 9. Error bars, s.e.m. P values were determined by ANOVA.

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

    (a) Quantitative real time PCR analysis of RAB genes in melanoma (SK-Mel-), breast cancer (MCF7, MDA-MB-231 and SkBr3) and pancreatic adenocarcinoma (AsPc1) human cell lines. Red denotes high (at least twofold), white denotes intermediate (less than twofold and >1.5-fold) and blue denotes low (≤1.5-fold) RAB expression in melanoma relative to breast cancer and pancreatic cell lines. (b) Measurement of the total amount of protein in the exosomes secreted per million human melanoma cells in culture. (c) Quantitative real time PCR analysis of Rab27a expression after shRNA knockdown of Rab27a in the B16-F10 and SK-Mel-28 cell lines. (d) Measurement of exosome protein per million cells after shRNA knockdown of Rab27a in the 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-F10shScramble and B16-F10shRab27a cell lines. Numbers in parentheses below indicate the spots analyzed for the membrane (right image). (f) Analysis of primary tumor growth and metastasis in B16-F10shScramble, B16-F10shRab27a and SK-Mel-28 cell lines subcutaneously injected into the flanks of C57BL/6 (B16-F10shScramble and B16-F10shRab27a) and nonobese diabetic severe combined immunodeficient mice (SK-Mel-28). Metastases were macroscopically counted (B16-F10) or quantified by quantitative real time PCR for mCherry (SK-Mel-28). n = 5 mice per group. Error bars, s.e.m. ***P < 0.001 by ANOVA. (g) Analysis of BMDCs (GFP+, green) and tumor cells (mCherry, red) in B16-F10shScramble and B16-F10shRab27a primary tumors (top, scale bar, 200 μm) and lungs (bottom, scale bar, 50 μm). Quantification of the metastatic area and the total number of BMDCs is shown on the right. n = 5 mice per group. Error bars, s.e.m. P values were determined by ANOVA.

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Primary accessions

Gene Expression Omnibus

Change history

Corrected online 05 October 2016
In the version of this article initially published, the upper and middle panels on the left-hand side in Figure 4a were interchanged, and the GAPDH loading panel in Supplementary Figure 6 was incorrect owing to an error in assembling the figures. The original western blot was rescanned to generate the correct GAPDH loading panel for Supplementary Figure 6. A new western blot was scanned to generate the bottom panel on the lefthand side in Figure 4a because the authors could not find the original western blot of the GAPDH loading panel. The errors have been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, Cell and Developmental Biology, Weill Cornell Medical College, New York, New York, USA.

    • Héctor Peinado,
    • Irina Matei,
    • Bruno Costa-Silva,
    • Caitlin Williams,
    • Guillermo García-Santos,
    • Ayuko Nitadori-Hoshino,
    • Karen Badal &
    • David Lyden
  2. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

    • Maša Alečković,
    • Benjamin A Garcia &
    • Yibin Kang
  3. Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

    • Simon Lavotshkin
  4. International Center for Research and Education, A.C. Camargo Hospital, São Paulo, Brazil.

    • Bruno Costa-Silva &
    • Vilma R Martins
  5. Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas (CSIC)-UAM, IdiPAZ (Instituto de Investigación Sanitaria La Paz) & Fundación MD Anderson Cancer Center, Madrid, Spain.

    • Gema Moreno-Bueno &
    • Marta Hergueta-Redondo
  6. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Cyrus M Ghajar
  7. Department of Neurosurgery, Weill Cornell Medical College, New York, New York, USA.

    • Caitlin Hoffman
  8. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Margaret K Callahan,
    • Jedd D Wolchok,
    • Paul B Chapman &
    • Jacqueline Bromberg
  9. Department of Immunology, Ludwig Center for Cancer Immunotherapy, Sloan-Kettering Institute, New York, New York, USA.

    • Jianda Yuan &
    • Jedd D Wolchok
  10. Exosome Diagnostics Inc., New York, New York, USA.

    • Johan Skog
  11. Pediatric Oncology Branch, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.

    • Rosandra N Kaplan
  12. Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Mary S Brady
  13. Weill Cornell Medical College, New York, New York, USA.

    • Jedd D Wolchok,
    • Paul B Chapman &
    • Jacqueline Bromberg
  14. Genomic Instability and Tumor Progression Program, Cancer Institute of New Jersey, New Brunswick, New Jersey, USA.

    • Yibin Kang
  15. Champalimaud Metastasis Programme, Lisbon, Portugal.

    • Yibin Kang &
    • David Lyden
  16. Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • David Lyden

Contributions

H.P. developed the hypothesis, designed the experimental approach, performed the experimental work, analyzed the data, coordinated the project and wrote the manuscript. S.L. conducted experimental work. I.M. performed flow cytometry studies and analysis. B.C.-S. conducted mouse work and proteomic characterization of exosomes. G.M.-B. conducted gene expression studies and analysis of microarray data. M.H.-R. conducted mouse work and bone marrow education studies. C.W. conducted mouse work and human studies. G.G.-S. developed bone marrow education assays. A.N.-H. and K.B. quantified exosomes in human plasma. M.A., B.A.G. and Y.K. performed mass spectrometry studies and contributed to data interpretation. C.H. obtained human blood specimens. M.K.C. and J.Y. contributed to the characterization of human plasma exosomes. J.S., R.N.K., V.R.M., M.S.B., J.D.W., P.B.C. and C.M.G. discussed the hypothesis and contributed to data interpretation and experimental design. J.B. coordinated the project, interpreted data and wrote the manuscript. D.L. conceived the hypothesis, led the project, interpreted the data and wrote the manuscript.

Competing financial interests

J.S. is an inventor on patent applications using microvesicle nucleic acid for diagnostics, which has been licensed to Exosome Diagnostics, Inc. He holds equity in and is an employee of that company.

Corresponding authors

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