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Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy

Abstract

Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell–derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.

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Figure 1: Human glioblastoma cells produce abundant microvesicles, which can be analyzed by μNMR.
Figure 2: μNMR assay for microvesicle detection.
Figure 3: Protein typing of GBM-derived microvesicles from cell lines and patient samples.
Figure 4: Effects of geldanamycin treatment on T103 GBM model.
Figure 5: Analysis of circulating microvesicles in GBM mice and human patients undergoing treatment.

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References

  1. Maheswaran, S. et al. Detection of mutations in EGFR in circulating lung-cancer cells. N. Engl. J. Med. 359, 366–377 (2008).

    Article  CAS  Google Scholar 

  2. Kulasingam, V., Pavlou, M.P. & Diamandis, E.P. Integrating high-throughput technologies in the quest for effective biomarkers for ovarian cancer. Nat. Rev. Cancer 10, 371–378 (2010).

    Article  CAS  Google Scholar 

  3. Simpson, R.J., Lim, J.W., Moritz, R.L. & Mathivanan, S. Exosomes: proteomic insights and diagnostic potential. Expert Rev. Proteomics 6, 267–283 (2009).

    Article  CAS  Google Scholar 

  4. Théry, C., Ostrowski, M. & Segura, E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9, 581–593 (2009).

    Article  Google Scholar 

  5. Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476 (2008).

    Article  CAS  Google Scholar 

  6. Al-Nedawi, K. et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat. Cell Biol. 10, 619–624 (2008).

    Article  CAS  Google Scholar 

  7. Simons, M. & Raposo, G. Exosomes—vesicular carriers for intercellular communication. Curr. Opin. Cell Biol. 21, 575–581 (2009).

    Article  CAS  Google Scholar 

  8. Lee, T.H. et al. Microvesicles as mediators of intercellular communication in cancer—the emerging science of cellular 'debris'. Semin. Immunopathol. 33, 455–467 (2011).

    Article  Google Scholar 

  9. Cocucci, E., Racchetti, G. & Meldolesi, J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 19, 43–51 (2009).

    Article  CAS  Google Scholar 

  10. Graner, M.W. et al. Proteomic and immunologic analyses of brain tumor exosomes. FASEB J. 23, 1541–1557 (2009).

    Article  CAS  Google Scholar 

  11. Sanderson, M.P. et al. Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion. J. Cell. Biochem. 103, 1783–1797 (2008).

    Article  CAS  Google Scholar 

  12. Balaj, L. et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2, 180 (2011).

    Article  Google Scholar 

  13. Lee, H., Sun, E., Ham, D. & Weissleder, R. Chip-NMR biosensor for detection and molecular analysis of cells. Nat. Med. 14, 869–874 (2008).

    Article  Google Scholar 

  14. Issadore, D. et al. Miniature magnetic resonance system for point-of-care diagnostics. Lab Chip 11, 2282–2287 (2011).

    Article  CAS  Google Scholar 

  15. Haun, J.B. et al. Micro-NMR for rapid molecular analysis of human tumor samples. Sci. Transl. Med. 3, 71ra16 (2011).

    Article  Google Scholar 

  16. Théry, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579 (2002).

    Article  Google Scholar 

  17. Haun, J.B., Devaraj, N.K., Hilderbrand, S.A., Lee, H. & Weissleder, R. Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection. Nat. Nanotechnol. 5, 660–665 (2010).

    Article  CAS  Google Scholar 

  18. Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 30, 3.22 (2006).

    Article  Google Scholar 

  19. Fleming, T.P. et al. Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors. Cancer Res. 52, 4550–4553 (1992).

    CAS  PubMed  Google Scholar 

  20. Mishima, K. et al. Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression. Acta Neuropathol. 111, 483–488 (2006).

    Article  CAS  Google Scholar 

  21. Wykosky, J., Gibo, D.M., Stanton, C. & Debinski, W. EphA2 as a novel molecular marker and target in glioblastoma multiforme. Mol. Cancer Res. 3, 541–551 (2005).

    Article  CAS  Google Scholar 

  22. Parsons, D.W. et al. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807–1812 (2008).

    Article  CAS  Google Scholar 

  23. Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739–744 (2009).

    Article  CAS  Google Scholar 

  24. Italiano, J.E.J., Mairuhu, A.T. & Flaumenhaft, R. Clinical relevance of microparticles from platelets and megakaryocytes. Curr. Opin. Hematol. 17, 578–584 (2010).

    Article  Google Scholar 

  25. Pedersen, N.M. et al. Expression of epidermal growth factor receptor or ErbB3 facilitates geldanamycin-induced down-regulation of ErbB2. Mol. Cancer Res. 7, 275–284 (2009).

    Article  CAS  Google Scholar 

  26. Trepel, J., Mollapour, M., Giaccone, G. & Neckers, L. Targeting the dynamic HSP90 complex in cancer. Nat. Rev. Cancer 10, 537–549 (2010).

    Article  CAS  Google Scholar 

  27. Zhu, H. et al. The novel Hsp90 inhibitor NXD30001 induces tumor regression in a genetically engineered mouse model of glioblastoma multiforme. Mol. Cancer Ther. 9, 2618–2626 (2010).

    Article  CAS  Google Scholar 

  28. Zhu, H. et al. Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis. Proc. Natl. Acad. Sci. USA 106, 2712–2716 (2009).

    Article  CAS  Google Scholar 

  29. Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068 (2008).

  30. Verhaak, R.G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).

    Article  CAS  Google Scholar 

  31. Pedersen, M.W., Meltorn, M., Damstrup, L. & Poulsen, H.S. The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy. Ann. Oncol. 12, 745–760 (2001).

    Article  CAS  Google Scholar 

  32. Mellinghoff, I.K. et al. Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N. Engl. J. Med. 353, 2012–2024 (2005).

    Article  CAS  Google Scholar 

  33. Galbán, C.J. et al. The parametric response map is an imaging biomarker for early cancer treatment outcome. Nat. Med. 15, 572–576 (2009).

    Article  Google Scholar 

  34. Galbán, C.J. et al. Prospective analysis of parametric MRI biomarkers: Identification of early and distinct glioma response patterns not predicted by standard radiographic assessment. Clin. Cancer Res. 17, 4751–4760 (2011).

    Article  Google Scholar 

  35. Tsien, C. et al. Parametric response map as an imaging biomarker to distinguish progression from pseudoprogression in high-grade glioma. J. Clin. Oncol. 28, 2293–2299 (2010).

    Article  CAS  Google Scholar 

  36. Elkhaled, A. et al. Magnetic resonance of 2-hydroxyglutarate in IDH1-Mutated low-grade gliomas. Sci. Transl. Med. 4, 116ra5 (2012).

    Article  Google Scholar 

  37. Wei, L.H. et al. Changes in tumor metabolism as readout for mammalian target of rapamycin kinase inhibition by rapamycin in glioblastoma. Clin. Cancer Res. 14, 3416–3426 (2008).

    Article  CAS  Google Scholar 

  38. Yoon, T.J., Lee, H., Shao, H. & Weissleder, R. Highly magnetic core-shell nanoparticles with a unique magnetization mechanism. Angew. Chem. Int. Edn Engl. 50, 4663–4666 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Reiner (Massachusetts General Hospital (MGH)) for preparing TCO, N. Sergeyev (MGH) for synthesizing MNPs, S. Hilderbrand (MGH) for synthesizing reactive TZ, M. Pittet (MGH) for LNZ308 cells and T. Chan (Memorial Sloan-Kettering Cancer Center) for SkMG3 cells, as well as M. Liong and A. Ghazani for assay assistance, B. Marinelli for μNMR measurements, C. Min for software implementation, M. McKee for transmission electron microscopy, J. Skog for advice on NTA measurements, L. Zhu and S. Sivaraman for technical assistance and Y. Fisher-Jeffes for critical reading of the manuscript. Special thanks to C. Castro, J. Carlson and clinical colleagues for many helpful discussions. This work was supported in part by NIH grants U54CA151884, R01EB010011, R01EB004626, P01CA069246, P50CA86355, U01CA141556, U24CA092782 and R21CA14122; H.S. received a BS-PhD National Science Scholarship awarded by the Singapore Agency for Science, Technology and Research; A.C. received an American Cancer Society Research Scholar Award 117409.

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H.S., R.W. and H.L. designed the study. H.S., J.C., L.B. and H.L. performed the experiments. H.S., J.C., R.W. and H.L. analyzed the data and wrote the manuscript. A.C. generated the mouse T103 model. D.D.B. recommended GBM biomarkers and generated the EGFRvIII-specific antibody. B.S.C., F.H.H. and X.O.B. coordinated the clinical study and analyzed the results.

Corresponding authors

Correspondence to Ralph Weissleder or Hakho Lee.

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The authors declare no competing financial interests.

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Supplementary Methods, Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 2579 kb)

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Shao, H., Chung, J., Balaj, L. et al. Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat Med 18, 1835–1840 (2012). https://doi.org/10.1038/nm.2994

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