Advertisement

Journal of Neuro-Oncology

, Volume 117, Issue 1, pp 15–24 | Cite as

Detection of primary cilia in human glioblastoma

  • Matthew R. SarkisianEmail author
  • Dorit Siebzehnrubl
  • Lan Hoang-Minh
  • Loic Deleyrolle
  • Daniel J. Silver
  • Florian A. Siebzehnrubl
  • Sarah M. Guadiana
  • Gayathri Srivinasan
  • Susan Semple-Rowland
  • Jeffrey K. Harrison
  • Dennis A. Steindler
  • Brent A. Reynolds
Laboratory Investigation

Abstract

Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies.

Keywords

Glioma Cilium Intraflagellar transport Cancer 

Notes

Acknowledgments

We would like to thank the UF COM Core EM Facility and R. Martuscello for technical assistance, and T. Caspary for the rabbit Arl13b antibody. We would also like to thank B. Frentzen and R. McTiernan of the Florida Center for Brain Tumor Research for providing us with tumor samples. This work was supported, in part, by funds from the McKnight Brain Research Foundation and the Evelyn F. and William L. McKnight Brain Institute at the University of Florida (to M.R.S., B.A.S and D.A.S.), an American Cancer Society Chris DiMarco Institutional Research Grant Junior Investigator Award (to M.R.S), and an American Cancer Society Research Scholar Grant (#RSG-13-031-01-DDC) (to M.R.S.). M.R.S. would like to dedicate this study to the memory of his younger brother, Andrew T. Sarkisian, who died in 2007 at the age of 28 from blood cancer.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

11060_2013_1340_MOESM1_ESM.tif (2.9 mb)
Supplementary material 1 (TIFF 2975 kb)

References

  1. 1.
    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996PubMedCrossRefGoogle Scholar
  2. 2.
    Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H (2012) The brain tumor microenvironment. Glia 60:502–514PubMedCrossRefGoogle Scholar
  3. 3.
    Hjelmeland AB, Lathia JD, Sathornsumetee S, Rich JN (2011) Twisted tango: brain tumor neurovascular interactions. Nat Neurosci 14:1375–1381PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Siebzehnrubl FA, Reynolds BA, Vescovi A, Steindler DA, Deleyrolle LP (2011) The origins of glioma: E Pluribus Unum? Glia 59:1135–1147PubMedCrossRefGoogle Scholar
  5. 5.
    Breunig JJ, Sarkisian MR, Arellano JI, Morozov YM, Ayoub AE, Sojitra S, Wang B, Flavell RA, Rakic P, Town T (2008) Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc Natl Acad Sci USA 105:13127–13132PubMedCrossRefGoogle Scholar
  6. 6.
    Han YG, Spassky N, Romaguera-Ros M, Garcia-Verdugo JM, Aguilar A, Schneider-Maunoury S, Alvarez-Buylla A (2008) Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nat Neurosci 11:277–284PubMedCrossRefGoogle Scholar
  7. 7.
    Baudoin JP, Viou L, Launay PS, Luccardini C, Espeso Gil S, Kiyasova V, Irinopoulou T, Alvarez C, Rio JP, Boudier T, Lechaire JP, Kessaris N, Spassky N, Metin C (2012) Tangentially migrating neurons assemble a primary cilium that promotes their reorientation to the cortical plate. Neuron 76:1108–1122PubMedCrossRefGoogle Scholar
  8. 8.
    Higginbotham H, Eom TY, Mariani LE, Bachleda A, Hirt J, Gukassyan V, Cusack CL, Lai C, Caspary T, Anton ES (2012) Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex. Dev Cell 23:925–938PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Yoshimura K, Kawate T, Takeda S (2011) Signaling through the primary cilium affects glial cell survival under a stressed environment. Glia 59:333–344PubMedCrossRefGoogle Scholar
  10. 10.
    Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV (2003) Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426:83–87PubMedCrossRefGoogle Scholar
  11. 11.
    Rohatgi R, Milenkovic L, Scott MP (2007) Patched1 regulates hedgehog signaling at the primary cilium. Science 317:372–376PubMedCrossRefGoogle Scholar
  12. 12.
    Berbari NF, Lewis JS, Bishop GA, Askwith CC, Mykytyn K (2008) Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc Natl Acad Sci USA 105:4242–4246PubMedCrossRefGoogle Scholar
  13. 13.
    Chakravarthy B, Gaudet C, Menard M, Atkinson T, Chiarini A, Dal Pra I, Whitfield J (2010) The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells. Biochem Biophys Res Commun 401:458–462PubMedCrossRefGoogle Scholar
  14. 14.
    Schneider L, Clement CA, Teilmann SC, Pazour GJ, Hoffmann EK, Satir P, Christensen ST (2005) PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr Biol 15:1861–1866PubMedCrossRefGoogle Scholar
  15. 15.
    Ponten J, Macintyre EH (1968) Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:465–486PubMedCrossRefGoogle Scholar
  16. 16.
    Stein GH (1979) T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99:43–54PubMedCrossRefGoogle Scholar
  17. 17.
    Moser JJ, Fritzler MJ, Rattner JB (2009) Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells. BMC Cancer 9:448PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Barakat MT, Humke EW, Scott MP (2013) Kif3a is necessary for initiation and maintenance of medulloblastoma. Carcinogenesis 34:1382–1392PubMedCrossRefGoogle Scholar
  19. 19.
    Han YG, Kim HJ, Dlugosz AA, Ellison DW, Gilbertson RJ, Alvarez-Buylla A (2009) Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med 15:1062–1065PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Wong SY, Seol AD, So PL, Ermilov AN, Bichakjian CK, Epstein EH Jr, Dlugosz AA, Reiter JF (2009) Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. Nat Med 15:1055–1061PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Deleyrolle LP, Harding A, Cato K, Siebzehnrubl FA, Rahman M, Azari H, Olson S, Gabrielli B, Osborne G, Vescovi A, Reynolds BA (2011) Evidence for label-retaining tumour-initiating cells in human glioblastoma. Brain 134:1331–1343PubMedCrossRefGoogle Scholar
  23. 23.
    Hothi P, Martins TJ, Chen L, Deleyrolle L, Yoon JG, Reynolds B, Foltz G (2012) High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells. Oncotarget 3:1124–1136PubMedGoogle Scholar
  24. 24.
    Aldaz H, Rice LM, Stearns T, Agard DA (2005) Insights into microtubule nucleation from the crystal structure of human gamma-tubulin. Nature 435:523–527PubMedCrossRefGoogle Scholar
  25. 25.
    Kim J, Lee JE, Heynen-Genel S, Suyama E, Ono K, Lee K, Ideker T, Aza-Blanc P, Gleeson JG (2010) Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464:1048–1051PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci USA 90:5519–5523PubMedCrossRefGoogle Scholar
  27. 27.
    Ishikawa H, Marshall WF (2011) Ciliogenesis: building the cell’s antenna. Nature Rev Mol Cell Biol 12:222–234CrossRefGoogle Scholar
  28. 28.
    Santos N, Reiter JF (2008) Building it up and taking it down: the regulation of vertebrate ciliogenesis. Dev Dyn 237:1972–1981PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Caspary T, Larkins CE, Anderson KV (2007) The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell 12:767–778PubMedCrossRefGoogle Scholar
  30. 30.
    Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311–322PubMedCrossRefGoogle Scholar
  31. 31.
    Siebzehnrubl FA, Silver DJ, Tugertimur B, Deleyrolle LP, Siebzehnrubl D, Sarkisian MR, Devers KG, Yachnis AT, Kupper MD, Neal D, Nabilsi NH, Kladde MP, Suslov O, Brabletz S, Brabletz T, Reynolds BA, Steindler DA (2013) The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance. EMBO Mol Med 5:1196–1212PubMedCrossRefGoogle Scholar
  32. 32.
    Basten SG, Willekers S, Vermaat JS, Slaats GG, Voest EE, van Diest PJ, Giles RH (2013) Reduced cilia frequencies in human renal cell carcinomas versus neighboring parenchymal tissue. Cilia 2:2PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Kim J, Dabiri S, Seeley ES (2011) Primary cilium depletion typifies cutaneous melanoma in situ and malignant melanoma. PLoS One 6:e27410PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Seeley ES, Carriere C, Goetze T, Longnecker DS, Korc M (2009) Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia. Cancer Res 69:422–430PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    van Asselt SJ, de Vries EG, van Dullemen HM, Brouwers AH, Walenkamp AM, Giles RH, Links TP (2013) Pancreatic cyst development: insights from von Hippel-Lindau disease. Cilia 2:3PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Yuan K, Frolova N, Xie Y, Wang D, Cook L, Kwon YJ, Steg AD, Serra R, Frost AR (2010) Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues. J Histochem Cytochem 58:857–870PubMedCrossRefGoogle Scholar
  37. 37.
    Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, Park JK, Fine HA (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391–403PubMedCrossRefGoogle Scholar
  38. 38.
    Leask A, Obrietan K, Stearns T (1997) Synaptically coupled central nervous system neurons lack centrosomal gamma-tubulin. Neurosci Lett 229:17–20PubMedCrossRefGoogle Scholar
  39. 39.
    O’Connor AK, Malarkey EB, Berbari NF, Croyle MJ, Haycraft CJ, Bell PD, Hohenstein P, Kesterson RA, Yoder BK (2013) An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue. Cilia 2:8PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Fukushima N, Furuta D, Hidaka Y, Moriyama R, Tsujiuchi T (2009) Post-translational modifications of tubulin in the nervous system. J Neurochem 109:683–693PubMedCrossRefGoogle Scholar
  41. 41.
    Plotnikova OV, Golemis EA, Pugacheva EN (2008) Cell cycle-dependent ciliogenesis and cancer. Cancer Res 68:2058–2061PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Yang Y, Roine N, Makela TP (2013) CCRK depletion inhibits glioblastoma cell proliferation in a cilium-dependent manner. EMBO Rep 14:741–747PubMedCrossRefGoogle Scholar
  43. 43.
    Silver DJ, Siebzehnrubl FA, Schildts MJ, Yachnis AT, Smith GM, Smith AA, Scheffler B, Reynolds BA, Silver J, Steindler DA (2013) Chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment. J Neurosci 33:15603–15617PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Matthew R. Sarkisian
    • 1
    Email author
  • Dorit Siebzehnrubl
    • 1
  • Lan Hoang-Minh
    • 1
  • Loic Deleyrolle
    • 2
  • Daniel J. Silver
    • 2
  • Florian A. Siebzehnrubl
    • 2
  • Sarah M. Guadiana
    • 1
  • Gayathri Srivinasan
    • 1
  • Susan Semple-Rowland
    • 1
  • Jeffrey K. Harrison
    • 3
  • Dennis A. Steindler
    • 2
  • Brent A. Reynolds
    • 2
  1. 1.Department of NeuroscienceMcKnight Brain Institute, University of Florida College of MedicineGainesvilleUSA
  2. 2.Department of NeurosurgeryMcKnight Brain Institute, University of Florida College of MedicineGainesvilleUSA
  3. 3.Department of Pharmacology and TherapeuticsMcKnight Brain Institute, University of Florida College of MedicineGainesvilleUSA

Personalised recommendations