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Abstract

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

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  • 02 December 2015

    The x-axis labels in Fig. 2c were corrected.

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Acknowledgements

We thank C. Ruiz de Almodovar and H.-H. Gerdes for discussions and comments; P. Rübman, B. Kast, A. Habel, A. Tietz-Dalfuβ and M. Fischer for technical assistance; R. Hermann for help with vibratome slices; G. Eisele for providing the WJ cell line; P. Friedl for the Lifeact-YFP-construct and the IDH1R132H thick section staining protocol; H. Glimm for the pCCL.PPT.SFFV.MCS.IRES.eGFP.WPRE-vector backbone; and M. Splinter, M. Brand, C. Lang for help with radiation experiments. This work was funded by grants from the German Research Foundation (DFG, WI 1930/5-1 (F.W.) and Major Equipment Grant INST 114089/26-1 FUGG (F.W., W.W.)), an intramural grant from the DKFZ to F.W. and H.L., Heinrich F. C. Behr-Stipend to S. Weil. F.S. is a fellow of the Medical Faculty Heidelberg PostDoc-Program. The results published here are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

Author information

Author notes

    • Erik Jung
    • , Felix Sahm
    •  & Gergely Solecki

    These authors contributed equally to this work.

Affiliations

  1. Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany

    • Matthias Osswald
    • , Erik Jung
    • , Gergely Solecki
    • , Jonas Blaes
    • , Sophie Weil
    • , Benedikt Wiestler
    • , Mustafa Syed
    • , Lulu Huang
    • , Kianush Karimian Jazi
    • , Torsten Schmenger
    • , Dieter Lemke
    • , Miriam Gömmel
    • , Yunxiang Liao
    • , Michael Platten
    • , Wolfgang Wick
    •  & Frank Winkler
  2. Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    • Matthias Osswald
    • , Erik Jung
    • , Gergely Solecki
    • , Jonas Blaes
    • , Sophie Weil
    • , Benedikt Wiestler
    • , Mustafa Syed
    • , Lulu Huang
    • , Miriam Ratliff
    • , Kianush Karimian Jazi
    • , Torsten Schmenger
    • , Dieter Lemke
    • , Miriam Gömmel
    • , Yunxiang Liao
    • , Wolfgang Wick
    •  & Frank Winkler
  3. Department of Neuropathology, Institute of Pathology, Ruprecht-Karls University Heidelberg, INF 224, 69120 Heidelberg, Germany

    • Felix Sahm
    • , Stefan Pusch
    •  & Andreas von Deimling
  4. Clinical Cooperation Unit Neuropathology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), INF 224, 69120 Heidelberg, Germany

    • Felix Sahm
    • , Stefan Pusch
    •  & Andreas von Deimling
  5. Department of Functional Neuroanatomy, Institute of Anatomy and Cell Biology, Heidelberg University, INF 307, 69120 Heidelberg, Germany

    • Varun Venkataramani
    • , Heinz Horstmann
    •  & Thomas Kuner
  6. Department of Diagnostic and Interventional Neuroradiology, Klinikum rechts der Isar der Technischen Universität München, 81675 Munich, Germany

    • Benedikt Wiestler
  7. Neurosurgery Clinic, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany

    • Miriam Ratliff
  8. Department of Neuroradiology, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany

    • Felix T. Kurz
    •  & Sabine Heiland
  9. Department of Neurophysiology, Institute of Physiology, University of Würzburg, 97070 Würzburg, Germany

    • Martin Pauli
  10. Department of Medical Physics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    • Peter Häring
  11. Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany

    • Verena Herl
    •  & Christian Steinhäuser
  12. Light Microscopy Facility, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    • Damir Krunic
  13. Department of Translational Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    • Mostafa Jarahian
  14. Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway

    • Hrvoje Miletic
  15. Institute of Neurology, Medical University of Vienna, Vienna, Austria; Comprehensive Cancer Center, CNS Unit, Medical University of Vienna, 1090 Vienna, Austria

    • Anna S. Berghoff
  16. Tools For Bio-Imaging, Max-Planck-Institute of Neurobiology, 82152 Martinsried, Germany

    • Oliver Griesbeck
  17. Institute of Physiology II, Eberhard Karls University of Tübingen, 72074 Tübingen, Germany

    • Georgios Kalamakis
    •  & Olga Garaschuk
  18. Department of Medicine I, Medical University of Vienna, Vienna, Austria; Comprehensive Cancer Center, CNS Unit, Medical University of Vienna, 1090 Vienna, Austria

    • Matthias Preusser
  19. Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

    • Samuel Weiss
  20. Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4Z6, Canada

    • Samuel Weiss
  21. Clark Smith Brain Tumor Research Centre, Southern Alberta Cancer Research Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

    • Samuel Weiss
  22. Helmholtz Young Investigator Group, Normal and Neoplastic CNS Stem Cells, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), INF 280, 69120 Heidelberg, Germany

    • Haikun Liu
  23. Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    • Michael Platten
  24. CCU Molecular and Radiation Oncology, German Cancer Research Center (DKFZ), INF 280, 69120 Heidelberg, Germany

    • Peter E. Huber
  25. Department of Radiation Oncology, University Hospital Heidelberg, 69120 Heidelberg, Germany

    • Peter E. Huber

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Contributions

F.W., M.O. and W.W. were responsible for experimental design, data interpretation, and writing of the manuscript. M.O., E.J., S. Weil and Y.L. performed MPLSM experiments. F.S. and A.v.D. performed stainings and analyses of human glioma tissues. M.O., M.G., E.J., S. Weil performed cell culture and cranial window implantations. G.S. was responsible for quantification and analysis of the calcium data. T.K., H.H., V.V. provided electron microscopy data and corresponding analyses. B.W. performed the TCGA data analysis. F.T.K. and S.H. collected MRI data. J.B. and T.S., M.R. and K.K.J. performed cell culture experiments, S.P. and D.L. established and characterized cell lines. A.S.B., L.H. and M. Preusser conducted histological experiments. V.H. and C.S. constructed the rrl-CAG-lGC3 vector. O. Griesbeck, G.K. and O. Garaschuk constructed the Twitch-3 vector, and interpreted the calcium imaging data. M.S. performed analyses of thick human tumour slices. M. Pauli conducted electroporation experiments. P.H. and P.E.H. were responsible for radiation. D.K. performed analysis of image data and confocal image acquisition. M. Platten performed data interpretation. M.J. performed FACS sorting. H.M. and S. Weiss provided cell lines and interpreted data. H.L. provided the syngeneic tumour model.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Frank Winkler.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1 (gel source data) and Supplementary Figure 2 (MRI source data of animals not shown in the main or Extended Data Figures).  This file was updated on 11 November 2015 to correct old references.

Excel files

  1. 1.

    Supplementary Table

    This file contains Supplementary Table 1 which contains results of the differential gene expression analysis of (A) 1p/19q non-codeleted / IDH mutated (n=124) vs. codeleted / IDH mutated (n=70), and (B) 1p/19q non-codeleted / IDH wild-type (n=56) vs. codeleted / IDH mutated (n=70) grade II and III gliomas of the TCGA database. logFC, log fold-change; logCPM, log counts per million; FDR, false-discovery rate adjusted p value. A positive logFC value means relative overexpression in 1p/19q non-codeleted gliomas; a negative logFC value means relative overexpression in codeleted gliomas. GJA1: gene encoding connexin 43 protein. GAP43: gene encoding GAP-43 protein.

Videos

  1. 1.

    Long membrane tubes are extended from astrocytoma cells at the invasive front

    a) Brain invasion of S24-GFP GBMSCs that were implanted at day 0 into the mouse brain, and followed from day 13 to 62 by in vivo MPLSM in the same brain microregion. Note extension of ultra-long cellular protrusions at the invasive front. Green, S24-GFP cells; red, brain microvessels (TRITC dextran angiography). b) High-magnification time-lapse in vivo MPLSM of one astrocytoma cell reveals that protrusions arborize, and demonstrate a scanning behavior. The box shows a region where the two upper protrusions are extended, the lower is retracted.

  2. 2.

    Membrane tubes interconnect single astrocytoma cells to a multicellular network

    z-stacks of three different astrocytoma mouse models, to illustrate the 3D morphology of microtubes, and microtube-interconnected cellular networks: a) S24-GFP GBMSC xenografts after 60 days of growth in a mouse brain; b) T269-GFP GBMSC xenografts after 102 days of growth in a mouse brain, c) Genetic mouse model of astrocytoma, where a tumor cell subpopulation with stem-like properties is identified by GFP expression driven by the promotor of the nuclear receptor tailless (day 105 after tumor induction). Depth is given for focal planes. All images: in vivo MPLSM.

  3. 3.

    Intercellular membrane tubes in human astrocytoma

    Confocal microscopy (z-stack) of a IDH1-R132H immunohistochemical staining of a patients’ WHO III° astrocytoma.

  4. 4.

    Intercellular calcium waves (ICWs) involving TMs in astrocytomas

    Images were acquired by time-lapse in vivo MPLSM. Detection of tumor cell calcium transients in S24 GBMSCs by brain superfusion with the small molecule calcium indicator Fluo4-AM (green). At the end, the RFP-expressing GBMSCs of this region are shown to demonstrate the cellular density and morphology of tumor cells.

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https://doi.org/10.1038/nature16071

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