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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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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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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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