Glioma-associated human endothelial cell-derived extracellular vesicles specifically promote the tumourigenicity of glioma stem cells via CD9


The perivascular niche in glioma is critical for the maintenance of glioma stem cells (GSCs), and tumour-endothelial cell (EC) communication impacts tumourigenesis in ways that are incompletely understood. Here, we show that glioma-associated human endothelial cells (GhECs), a main component of the perivascular niche, release extracellular vesicles (EVs) that increase GSC proliferation and tumour-sphere formation. GSCs treated with GhEC-EVs create a significantly greater tumour burden than do untreated GSCs in orthotopic xenografts. Mechanistic, analysis of EVs content identified CD9 as a mediator of the effects on GSCs. CD9 can activate the BMX/STAT3 signalling pathway in GSCs. Our results illuminate the tumour-supporting role of ECs by identifying that EC-derived EVs transfer of CD9 during intercellular communication, thereby enhancing the aggressiveness of glioblastoma by specifically maintaining GSCs.

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  1. 1.

    Yan K, Yang K, Rich JN. The evolving landscape of glioblastoma stem cells. Curr Opin Neurol. 2013;26:701–7.

  2. 2.

    Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756–60.

  3. 3.

    Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B, et al. A perivascular niche for brain tumor stem cells. Cancer Cell. 2007;11:69–82.

  4. 4.

    Galan-Moya EM, Le Guelte A, Lima Fernandes E, Thirant C, Dwyer J, Bidere N, et al. Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway. EMBO Rep. 2011;12:470–6.

  5. 5.

    Jeon HM, Kim SH, Jin X, Park JB, Kim SH, Joshi K, et al. Crosstalk between glioma-initiating cells and endothelial cells drives tumor progression. Cancer Res. 2014;74:4482–92.

  6. 6.

    Infanger DW, Cho Y, Lopez BS, Mohanan S, Liu SC, Gursel D, et al. Glioblastoma stem cells are regulated by interleukin-8 signaling in a tumoral perivascular niche. Cancer Res. 2013;73:7079–89.

  7. 7.

    Yan GN, Yang L, Lv YF, Shi Y, Shen LL, Yao XH, et al. Endothelial cells promote stem-like phenotype of glioma cells through activating the Hedgehog pathway. J Pathol. 2014;234:11–22.

  8. 8.

    Xu R, Greening DW, Zhu HJ, Takahashi N, Simpson RJ. Extracellular vesicle isolation and characterization: toward clinical application. J Clin Investig. 2016;126:1152–62.

  9. 9.

    Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015;4:27066.

  10. 10.

    Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteom. 2010;73:1907–20.

  11. 11.

    Atay S, Banskota S, Crow J, Sethi G, Rink L, Godwin AK. Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion. Proc Natl Acad Sci USA. 2014;111:711–6.

  12. 12.

    Di Modica M, Regondi V, Sandri M, Iorio MV, Zanetti A, Tagliabue E, et al. Breast cancer-secreted miR-939 downregulates VE-cadherin and destroys the barrier function of endothelial monolayers. Cancer Lett. 2017;384:94–100.

  13. 13.

    Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10:1470–6.

  14. 14.

    Leca J, Martinez S, Lac S, Nigri J, Secq V, Rubis M, et al. Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness. J Clin Investig. 2016;126:4140–56.

  15. 15.

    Boelens MC, Wu TJ, Nabet BY, Xu B, Qiu Y, Yoon T, et al. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways. Cell. 2014;159:499–513.

  16. 16.

    Bovy N, Blomme B, Freres P, Dederen S, Nivelles O, Lion M, et al. Endothelial exosomes contribute to the antitumor response during breast cancer neoadjuvant chemotherapy via microRNA transfer. Oncotarget. 2015;6:10253–66.

  17. 17.

    Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringner M, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci USA. 2013;110:7312–7.

  18. 18.

    Treps L, Perret R, Edmond S, Ricard D, Gavard J. Glioblastoma stem-like cells secrete the pro-angiogenic VEGF-A factor in extracellular vesicles. J Extracell Vesicles. 2017;6:1359479.

  19. 19.

    Treps L, Edmond S, Harford-Wright E, Galan-Moya EM, Schmitt A, Azzi S, et al. Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma. Oncogene. 2016;35:2615–23.

  20. 20.

    Figueroa J, Phillips LM, Shahar T, Hossain A, Gumin J, Kim H et al. Exosomes from glioma-associated mesenchymal stem cells increase the tumorigenicity of glioma stem-like cells via transfer of miR-1587. Cancer Res 2017;77:5808–5819.

  21. 21.

    Mahmoud AM, Wilkinson FL, McCarthy EM, Moreno-Martinez D, Langford-Smith A, Romero M, et al. Endothelial microparticles prevent lipid-induced endothelial damage via Akt/eNOS signaling and reduced oxidative stress. FASEB J. 2017;31:4636–48.

  22. 22.

    Valentijn KM, Valentijn JA, Jansen KA, Koster AJ. A new look at Weibel-Palade body structure in endothelial cells using electron tomography. J Struct Biol. 2008;161:447–58.

  23. 23.

    Zomer A, Maynard C, Verweij FJ, Kamermans A, Schafer R, Beerling E, et al. In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell. 2015;161:1046–57.

  24. 24.

    Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, et al. Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell Stem Cell. 2010;6:141–52.

  25. 25.

    Eyler CE, Wu Q, Yan K, MacSwords JM, Chandler-Militello D, Misuraca KL, et al. Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2. Cell. 2011;146:53–66.

  26. 26.

    Weyerbrock A, Osterberg N, Psarras N, Baumer B, Kogias E, Werres A, et al. JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas. Neurosurgery. 2012;70:497–510. discussion 510

  27. 27.

    Murayama Y, Shinomura Y, Oritani K, Miyagawa J, Yoshida H, Nishida M, et al. The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells. J Cell Physiol. 2008;216:135–43.

  28. 28.

    Kolesnikova TV, Kazarov AR, Lemieux ME, Lafleur MA, Kesari S, Kung AL, et al. Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo. Neoplasia. 2009;11:77–86.

  29. 29.

    Podergajs N, Motaln H, Rajcevic U, Verbovsek U, Korsic M, Obad N, et al. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells. Oncotarget. 2016;7:593–609.

  30. 30.

    Shi Y, Zhou W, Cheng L, Chen C, Huang Z, Fang X, et al. Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells. Cell Death Differ. 2017;24:167–80.

  31. 31.

    Bhuvanalakshmi G, Arfuso F, Millward M, Dharmarajan A, Warrier S. Secreted frizzled-related protein 4 inhibits glioma stem-like cells by reversing epithelial to mesenchymal transition, inducing apoptosis and decreasing cancer stem cell properties. PloS ONE. 2015;10:e0127517.

  32. 32.

    Bassett EA, Tokarew N, Allemano EA, Mazerolle C, Morin K, Mears AJ et al. Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation. eLife 2016;5:e16764.

  33. 33.

    Yamashita D, Kondo T, Ohue S, Takahashi H, Ishikawa M, Matoba R, et al. miR340 suppresses the stem-like cell function of glioma-initiating cells by targeting tissue plasminogen activator. Cancer Res. 2015;75:1123–33.

  34. 34.

    Solly F, Fish R, Simard B, Bolle N, Kruithof E, Polack B, et al. Tissue-type plasminogen activator has antiangiogenic properties without effect on tumor growth in a rat C6 glioma model. Cancer Gene Ther. 2008;15:685–92.

  35. 35.

    Yue S, Mu W, Erb U, Zoller M. The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding. Oncotarget. 2015;6:2366–84.

  36. 36.

    Wirsching HG, Krishnan S, Florea AM, Frei K, Krayenbuhl N, Hasenbach K, et al. Thymosin beta 4 gene silencing decreases stemness and invasiveness in glioblastoma. Brain. 2014;137:433–48.

  37. 37.

    Shi Y, Guryanova OA, Zhou W, Liu C, Huang Z, Fang X et al. Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance. Sci Transl Med 2018;10:6816.

  38. 38.

    Liu S, Sun J, Lan Q. Glioblastoma microvesicles promote endothelial cell proliferation through Akt/beta-catenin pathway. Int J Clin Exp Pathol. 2014;7:4857–66.

  39. 39.

    Kurachi M, Mikuni M, Ishizaki Y. Extracellular vesicles from vascular endothelial cells promote survival, proliferation and motility of oligodendrocyte precursor cells. PLoS ONE. 2016;11:e0159158.

  40. 40.

    Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor microenvironment. Glia. 2011;59:1169–80.

  41. 41.

    Filatova A, Acker T, Garvalov BK. The cancer stem cell niche(s): the crosstalk between glioma stem cells and their microenvironment. Biochim Biophys Acta. 2013;1830:2496–508.

  42. 42.

    Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol. 2005;7:122–33.

  43. 43.

    Osterberg N, Ferrara N, Vacher J, Gaedicke S, Niedermann G, Weyerbrock A, et al. Decrease of VEGF-A in myeloid cells attenuates glioma progression and prolongs survival in an experimental glioma model. Neuro Oncol. 2016;18:939–49.

  44. 44.

    Cherepanov SA, Cherepanova KI, Grinenko NF, Antonova OM, Chekhonin VP. Effect of hedgehog signaling pathway activation on proliferation of high-grade gliomas. Bull Exp Biol Med. 2016;161:674–8.

  45. 45.

    Cheng L, Bao S, Rich JN. Potential therapeutic implications of cancer stem cells in glioblastoma. Biochem Pharmacol. 2010;80:654–65.

  46. 46.

    Longo N, Yanez-Mo M, Mittelbrunn M, de la Rosa G, Munoz ML, Sanchez-Madrid F, et al. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells. Blood. 2001;98:3717–26.

  47. 47.

    Wang GP, Han XF. CD9 modulates proliferation of human glioblastoma cells via epidermal growth factor receptor signaling. Mol Med Rep. 2015;12:1381–6.

  48. 48.

    Miyake M, Nakano K, Itoi SI, Koh T, Taki T. Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer. Cancer Res. 1996;56:1244–9.

  49. 49.

    Huang CI, Kohno N, Ogawa E, Adachi M, Taki T, Miyake M. Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol. 1998;153:973–83.

  50. 50.

    Mori M, Mimori K, Shiraishi T, Haraguchi M, Ueo H, Barnard GF, et al. Motility related protein 1 (MRP1/CD9) expression in colon cancer. Clin Cancer Res. 1998;4:1507–10.

  51. 51.

    Hu PS, Xia QS, Wu F, Li DK, Qi YJ, Hu Y, et al. NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma. Oncogene. 2017;36:4706–18.

  52. 52.

    Chen C, Cao F, Bai L, Liu Y, Xie J, Wang W, et al. IKKbeta enforces a LIN28B/TCF7L2 positive feedback loop that promotes cancer cell stemness and metastasis. Cancer Res. 2015;75:1725–35.

  53. 53.

    Pan Y, Shu X, Sun L, Yu L, Sun L, Yang Z, et al. miR196a5p modulates gastric cancer stem cell characteristics by targeting Smad4. Int J Oncol. 2017;50:1965–76.

  54. 54.

    She X, Matsuno F, Harada N, Tsai H, Seon BK. Synergy between anti-endoglin (CD105) monoclonal antibodies and TGF-beta in suppression of growth of human endothelial cells. Int J Cancer. 2004;108:251–7.

  55. 55.

    Muller L, Mitsuhashi M, Simms P, Gooding WE, Whiteside TL. Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets. Sci Rep. 2016;6:20254.

  56. 56.

    Lombardo G, Dentelli P, Togliatto G, Rosso A, Gili M, Gallo S, et al. Activated Stat5 trafficking via endothelial cell-derived extracellular vesicles controls IL-3 pro-angiogenic paracrine action. Sci Rep. 2016;6:25689.

  57. 57.

    Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci USA. 2016;113:E968–977.

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We thank the Department of Neurosurgery of Tiantan Hospital for providing glioma surgical specimens and the CGGA database.


The National Key Research and Development Program of China (2016YFC0902500, 2016YFC0902502 and 2016YFA0100702), and National Sciences Foundation of China (31671316, 31670789), CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1–001, 2016-I2M-2–001, 2016-I2M-1–004, 2017-I2M-2–004, 2017-I2M-3–010 and 2017-I2M-1–004).

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Correspondence to Wei Han or Xiaozhong Peng.

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