Skip to main content
SpringerLink
Log in

High-Efficiency Transfection of Glioblastoma Cells and a Simple Spheroid Migration Assay

  • Protocol
  • First Online:
Book cover RNAi and Small Regulatory RNAs in Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1622))

Abstract

Despite international research efforts, patients with glioblastoma multiforme (GBM)—the most common malignant brain tumors in adults—exhibit a very unfavorable prognosis. Their aggressive local growth pattern and increased invasiveness, due to a high motility of the tumor cells, hamper treatment. However, the molecular mechanisms regulating glioblastoma cell migration are still elusive. Here, we describe the combination of a highly efficient cell transfection by Nucleofection® technology and the generation of spheroids from these transfected glioblastoma cell lines. Nucleofection allows the manipulation of protein expression by overexpression and siRNA mediated protein knockdown. Transfection efficiencies >70% can be achieved with some GBM cell lines. Transfected neurospheres then can be used for migration assays (as described here in detail) and a multitude of other functional assays. In comparison to monolayer cultures, the advantage of spheroids is their resemblance with organized tissue in combination with the accuracy of in vitro methodology and marked experimental flexibility.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Reifenberger G, Collins VP (2004) Pathology and molecular genetics of astrocytic gliomas. J Mol Med 82:656–670

    Article  CAS  PubMed  Google Scholar 

  2. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiation Oncology Groups, and National Cancer Institute of Canada Clinical Trials Group (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466

    Article  CAS  PubMed  Google Scholar 

  3. Weller M, van den Bent M, Hopkins K, Tonn JC, Stupp R, Falini A, Cohen-Jonathan-Moyal E, Frappaz D, Henriksson R, Balana C, Chinot O, Ram Z, Reifenberger G, Soffietti R, Wick W, European Association for Neuro-Oncology (EANO) Task Force on Malignant Glioma (2014) EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol 15:e395–e403

    Article  PubMed  Google Scholar 

  4. Demuth T, Berens ME (2004) Molecular mechanisms of glioma cell migration and invasion. J Neuroncol 70:217–228

    Article  Google Scholar 

  5. Sahm F, Capper D, Jeibmann A, Habel A, Paulus W, Troost D, von Deimling A (2012) Addressing diffuse glioma as a systemic brain disease with single-cell analysis. Arch Neurol 69:523–526

    Article  PubMed  Google Scholar 

  6. Stummer W, Reulen H-J, Meinel T, Pichlmeier U, Schumacher W, Tonn J-C, Rohde V, Oppel F, Turowski B, Woiciechowsky C, Franz K, Pietsch T, ALA-Glioma Study Group (2008) Extent of resection and survival in Glioblastoma multiforme: identification of and adjustment for bias. Neurosurgery 62:564–576

    Article  PubMed  Google Scholar 

  7. Pilkington GJ, Bjerkvig R, De Ridder L, Kaaijk P (1997) In vitro and in vivo models for the study of brain tumour invasion. Anticancer Res 17:4107–4109

    CAS  PubMed  Google Scholar 

  8. Martin WMC, McNally NJ (1980) Cytotoxicity of Adriamycin to tumour cells in vivo and in vitro. Br J Cancer 42:881–889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nederman T (1984) Effects of Vinblastine and 5-Fluorouracil on human glioma and thyroid cancer monolayers and spheroids. Cancer Res 44:254–258

    CAS  PubMed  Google Scholar 

  10. Yuhas JM, Li AP, Martinez AO, Ladman AJ (1977) A simplified method for production and growth of multicellular tumor spheroids. Cancer Res 37:3639–3643

    CAS  PubMed  Google Scholar 

  11. Sutherland RM, McCredie JA, Inch WR (1971) Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 46:113–120

    CAS  PubMed  Google Scholar 

  12. Sutherland RM (1980) The multicellular spheroid system as a tumor model for studies of radiation sensitizers. Pharmacol Ther 8:105–123

    Article  CAS  Google Scholar 

  13. Engebraaten O, Bjerkvig R, Berens ME (1991) Effect of Alkyl-lysophospholipid on Glioblastoma cell invasion into fetal rat brain tissue in vitro. Cancer Res 51:1713–1719

    CAS  PubMed  Google Scholar 

  14. Dilnawaz F, Sahoo SK (2013) Enhanced accumulation of curcumin and temozolomide loaded magnetic nanoparticles executes profound cytotoxic effect in glioblastoma spheroid model. Eur J Pharm Biopharm 85:452–462

    Article  CAS  PubMed  Google Scholar 

  15. Escoffre JM, Novell A, de Smet M, Bouakaz A (2013) Focused ultrasound mediated drug delivery from temperature-sensitive liposomes: in-vitro characterization and validation. Phys Med Biol 58:8135–8151

    Article  PubMed  Google Scholar 

  16. Farrell CL, Stewart PA, Del Maestro RF (1987) A new glioma model in rat: the C6 spheroid implantation technique permeability and vascular characterization. J Neurooncol 4:403–415

    Article  CAS  PubMed  Google Scholar 

  17. Goldbrunner RH, Wagner S, Roosen K, Tonn JC (2000) Models for assessment of angiogenesis in gliomas. Neuro Oncol 50:53–62

    Article  CAS  Google Scholar 

  18. da Cruz MT, Simoes S, de Lima MC (2004) Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons. Exp Neurol 187:65–75

    Article  PubMed  Google Scholar 

  19. Hagemann C, Meyer C, Stojic J, Eicker S, Gerngras S, Kühnel S, Roosen K, Vince GH (2006) High efficiency transfection of glioma cell lines and primary cells for overexpression and RNAi experiments. J Neurosci Methods 156:194–202

    Article  CAS  PubMed  Google Scholar 

  20. Christine R, Siebenkotten G (2000) Ausnutzung zelleigener Transportsysteme zum Transfer von Nukleinsäuren durch die Kernhülle. Patent-Offenlegungsschrift DE 19933939 A1, German Patent and Trade Mark Office

    Google Scholar 

  21. Rothmann-Cosic K, Wessendorf H, Helfrich J, Thiel C, Riemen G, Brosterhus H, Müller-Hartmann H, Weigel M, Lorbach E, Nix M, Siebenkotten G (2002) Buffer solution for electroporation and a method comprising the use of the same. Europäische Patentschrift EP 1390518 B1, European Patent Office

    Google Scholar 

  22. Pontén J, Macintyre EH (1968) Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:465–486

    Article  PubMed  Google Scholar 

  23. Beckman G, Beckman L, Pontén J, Westermark B (1971) G-6-PD and PGM phenotypes of 16 continuous human tumor cell lines. Evidence against cross-contamination and contamination by HeLa cells. Hum Hered 21:238–241

    Article  CAS  PubMed  Google Scholar 

  24. Kruse CA, Varella-Garcia M, Kleinschmidt-Demasters BK, Owens GC, Spector EB, Fakhrai H, Savelieva E, Liang BC (1998) Receptor expression, cytogenetic, and molecular analysis of six continuous human glioma cell lines. In Vitro Cell Dev Biol Anim 34:455–462

    Article  CAS  PubMed  Google Scholar 

  25. Westermark B, Pontén J, Hugosson R (1973) Determination for the establishment of permanent tissue culture from human gliomas. Acta Pathol Microbiol Scand 81:791–805

    CAS  Google Scholar 

  26. Bigner DD, Bigner SH, Pontén J, Westermark B, Mahaley MS, Ruoslahti E, Herschman H, Eng LF, Wikstrand CJ (1981) Heterogeneity of genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas. J Neuropathol Exp Neurol 40:201–229

    Article  CAS  PubMed  Google Scholar 

  27. Gross JL, Behrens DL, Mullins DE, Kornblith PL, Dexter DL (1988) Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res 48:291–296

    CAS  PubMed  Google Scholar 

  28. Akslen LA, Andersen KJ, Bjerkvig R (1988) Characteristics of human and rat glioma cells grown in a defined medium. Anticancer Res 8:797–803

    CAS  PubMed  Google Scholar 

  29. Gracia E, Fischer U, ElKahloun A, Trent JM, Meese E, Meltzer PS (1996) Isolation of genes amplified in human cancers by microdissection mediated cDNA capture. Hum Mol Genet 5:595–600

    Article  CAS  PubMed  Google Scholar 

  30. Timmerman D, Yeung CM (2014) Identity confusion of glioma cell lines. Gene 536:221–222

    Article  Google Scholar 

  31. Stepanenko AA, Kavsan VM (2014) Karyotypically distinct U251, U373 and SNB19 glioma cell lines are of the same origin but have different drug treatment sensitivities. Gene 540:263–265

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to Siglinde Kühnel and Elisabeth Karl for technical assistance. We also thank Alexandra Schock for the data she produced during an internship in the laboratory.

Author information

Authors and Affiliations

  1. Tumorbiology Laboratory, Department of Neurosurgery, University of Würzburg, Josef-Schneider-Str. 11, 97080, Würzburg, Germany

    Carsten Hagemann, Diana Amend, Almuth F. Kessler, Thomas Linsenmann, Ralf-Ingo Ernestus & Mario Löhr

Authors
  1. Carsten Hagemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Diana Amend
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Almuth F. Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Linsenmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ralf-Ingo Ernestus
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mario Löhr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carsten Hagemann .

Editor information

Editors and Affiliations

  1. Department of Biology, East Carolina University, Greenville, North Carolina, USA

    Baohong Zhang

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Hagemann, C., Amend, D., Kessler, A.F., Linsenmann, T., Ernestus, RI., Löhr, M. (2017). High-Efficiency Transfection of Glioblastoma Cells and a Simple Spheroid Migration Assay. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7108-4_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7106-0

  • Online ISBN: 978-1-4939-7108-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation