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Establishment and Culture of Patient-Derived Primary Medulloblastoma Cell Lines

  • Sara Badodi
  • Silvia MarinoEmail author
  • Loredana Guglielmi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1869)

Abstract

Established cell lines have been extensively used in cancer research. They are easy to obtain and expand and are composed of a relatively uniform population of cells. When experimental conditions are kept standard, these cells allow a high reproducibility of experimental findings from independent research groups. However, because these cell lines have been propagated in culture for decades, additional genetic lesions may be acquired leading to modification of their characteristics as compared to the original tumor. Primary cultures represent a valid alternative. Here, we describe standardized protocols to establish medulloblastoma (MB) patient-derived primary cultures from fresh tumor samples. MB primary cells grow as an adherent culture on a laminin coating and can be propagated in vitro for a limited number of passages, therefore reducing the chances to accumulate molecular alterations compared to long-term cultures. Consequently, they better resemble the original tumor both in terms of biological behavior and molecular characteristics. Low-passage MB primary cells can be used as an in vitro model for biochemical studies and functional assays, representing a useful tool to dissect the contribution of molecular pathways to MB pathogenesis. They can also represent a useful screening tool for potential therapeutic agents in preclinical studies.

Key words

Medulloblastoma Cell lines Primary cultures Molecular classification Adherent Laminin Epidermal growth factor Fibroblast growth factor 

References

  1. 1.
    Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, Bouffet E, Clifford SC, Hawkins CE, French P, Rutka JT, Pfister S, Taylor MD (2011) Medulloblastoma comprises four distinct molecular variants. J Clin Oncol 29(11):1408–1414.  https://doi.org/10.1200/JCO.2009.27.4324CrossRefPubMedGoogle Scholar
  2. 2.
    Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, Ellison DW, Lichter P, Gilbertson RJ, Pomeroy SL, Kool M, Pfister SM (2012) Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 123(4):465–472.  https://doi.org/10.1007/s00401-011-0922-zCrossRefPubMedGoogle Scholar
  3. 3.
    Thompson MC, Fuller C, Hogg TL, Dalton J, Finkelstein D, Lau CC, Chintagumpala M, Adesina A, Ashley DM, Kellie SJ, Taylor MD, Curran T, Gajjar A, Gilbertson RJ (2006) Genomics identifies medulloblastoma subgroups that are enriched for specific genetic alterations. J Clin Oncol 24(12):1924–1931.  https://doi.org/10.1200/JCO.2005.04.4974CrossRefPubMedGoogle Scholar
  4. 4.
    Clifford SC, Lusher ME, Lindsey JC, Langdon JA, Gilbertson RJ, Straughton D, Ellison DW (2006) Wnt/wingless pathway activation and chromosome 6 loss characterize a distinct molecular sub-group of medulloblastomas associated with a favorable prognosis. Cell Cycle 5(22):2666–2670.  https://doi.org/10.4161/cc.5.22.3446CrossRefPubMedGoogle Scholar
  5. 5.
    Taylor MD, Liu L, Raffel C, Hui CC, Mainprize TG, Zhang X, Agatep R, Chiappa S, Gao L, Lowrance A, Hao A, Goldstein AM, Stavrou T, Scherer SW, Dura WT, Wainwright B, Squire JA, Rutka JT, Hogg D (2002) Mutations in SUFU predispose to medulloblastoma. Nat Genet 31(3):306–310.  https://doi.org/10.1038/ng916CrossRefPubMedGoogle Scholar
  6. 6.
    Kool M, Jones DT, Jager N, Northcott PA, Pugh TJ, Hovestadt V, Piro RM, Esparza LA, Markant SL, Remke M, Milde T, Bourdeaut F, Ryzhova M, Sturm D, Pfaff E, Stark S, Hutter S, Seker-Cin H, Johann P, Bender S, Schmidt C, Rausch T, Shih D, Reimand J, Sieber L, Wittmann A, Linke L, Witt H, Weber UD, Zapatka M, Konig R, Beroukhim R, Bergthold G, van Sluis P, Volckmann R, Koster J, Versteeg R, Schmidt S, Wolf S, Lawerenz C, Bartholomae CC, von Kalle C, Unterberg A, Herold-Mende C, Hofer S, Kulozik AE, von Deimling A, Scheurlen W, Felsberg J, Reifenberger G, Hasselblatt M, Crawford JR, Grant GA, Jabado N, Perry A, Cowdrey C, Croul S, Zadeh G, Korbel JO, Doz F, Delattre O, Bader GD, McCabe MG, Collins VP, Kieran MW, Cho YJ, Pomeroy SL, Witt O, Brors B, Taylor MD, Schuller U, Korshunov A, Eils R, Wechsler-Reya RJ, Lichter P, Pfister SM, Project IPT (2014) Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell 25(3):393–405.  https://doi.org/10.1016/j.ccr.2014.02.004CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Roussel MF, Robinson GW (2013) Role of MYC in Medulloblastoma. Cold Spring Harb Perspect Med 3(11).  https://doi.org/10.1101/cshperspect.a014308CrossRefGoogle Scholar
  8. 8.
    Lin CY, Erkek S, Tong Y, Yin L, Federation AJ, Zapatka M, Haldipur P, Kawauchi D, Risch T, Warnatz HJ, Worst BC, Ju B, Orr BA, Zeid R, Polaski DR, Segura-Wang M, Waszak SM, Jones DT, Kool M, Hovestadt V, Buchhalter I, Sieber L, Johann P, Chavez L, Groschel S, Ryzhova M, Korshunov A, Chen W, Chizhikov VV, Millen KJ, Amstislavskiy V, Lehrach H, Yaspo ML, Eils R, Lichter P, Korbel JO, Pfister SM, Bradner JE, Northcott PA (2016) Active medulloblastoma enhancers reveal subgroup-specific cellular origins. Nature 530(7588):57–62.  https://doi.org/10.1038/nature16546CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cavalli FMG, Remke M, Rampasek L, Peacock J, Shih DJH, Luu B, Garzia L, Torchia J, Nor C, Morrissy AS, Agnihotri S, Thompson YY, Kuzan-Fischer CM, Farooq H, Isaev K, Daniels C, Cho B-K, Kim S-K, Wang K-C, Lee JY, Grajkowska WA, Perek-Polnik M, Vasiljevic A, Faure-Conter C, Jouvet A, Giannini C, Nageswara Rao AA, Li KKW, Ng H-K, Eberhart CG, Pollack IF, Hamilton RL, Gillespie GY, Olson JM, Leary S, Weiss WA, Lach B, Chambless LB, Thompson RC, Cooper MK, Vibhakar R, Hauser P, van Veelen M-LC, Kros JM, French PJ, Ra YS, Kumabe T, López-Aguilar E, Zitterbart K, Sterba J, Finocchiaro G, Massimino M, Van Meir EG, Osuka S, Shofuda T, Klekner A, Zollo M, Leonard JR, Rubin JB, Jabado N, Albrecht S, Mora J, Van Meter TE, Jung S, Moore AS, Hallahan AR, Chan JA, Tirapelli DPC, Carlotti CG, Fouladi M, Pimentel J, Faria CC, Saad AG, Massimi L, Liau LM, Wheeler H, Nakamura H, Elbabaa SK, Perezpeña-Diazconti M, Chico Ponce de León F, Robinson S, Zapotocky M, Lassaletta A, Huang A, Hawkins CE, Tabori U, Bouffet E, Bartels U, Dirks PB, Rutka JT, Bader GD, Reimand J, Goldenberg A, Ramaswamy V, Taylor MD (2017) Intertumoral heterogeneity within medulloblastoma subgroups. Cancer Cell 31(6):737–754.e6CrossRefGoogle Scholar
  10. 10.
    Schwalbe EC, Lindsey JC, Nakjang S, Crosier S, Smith AJ, Hicks D, Rafiee G, Hill RM, Iliasova A, Stone T, Pizer B, Michalski A, Joshi A, Wharton SB, Jacques TS, Bailey S, Williamson D, Clifford SC (2017) Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study. Lancet Oncol 18(7):958–971CrossRefGoogle Scholar
  11. 11.
    Gottardo NG, Hansford JR, McGlade JP, Alvaro F, Ashley DM, Bailey S, Baker DL, Bourdeaut F, Cho YJ, Clay M, Clifford SC, Cohn RJ, Cole CH, Dallas PB, Downie P, Doz F, Ellison DW, Endersby R, Fisher PG, Hassall T, Heath JA, Hii HL, Jones DT, Junckerstorff R, Kellie S, Kool M, Kotecha RS, Lichter P, Laughton SJ, Lee S, McCowage G, Northcott PA, Olson JM, Packer RJ, Pfister SM, Pietsch T, Pizer B, Pomeroy SL, Remke M, Robinson GW, Rutkowski S, Schoep T, Shelat AA, Stewart CF, Sullivan M, Taylor MD, Wainwright B, Walwyn T, Weiss WA, Williamson D, Gajjar A (2014) Medulloblastoma down under 2013: a report from the third annual meeting of the international Medulloblastoma working group. Acta Neuropathol 127(2):189–201.  https://doi.org/10.1007/s00401-013-1213-7CrossRefPubMedGoogle Scholar
  12. 12.
    Ivanov DP, Coyle B, Walker DA, Grabowska AM (2016) In vitro models of medulloblastoma: choosing the right tool for the job. J Biotechnol 236:10–25.  https://doi.org/10.1016/j.jbiotec.2016.07.028CrossRefPubMedGoogle Scholar
  13. 13.
    Shu Q, Wong KK, Su JM, Adesina AM, Yu LT, Tsang YT, Antalffy BC, Baxter P, Perlaky L, Yang J, Dauser RC, Chintagumpala M, Blaney SM, Lau CC, Li XN (2008) Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma. Stem Cells 26(6):1414–1424.  https://doi.org/10.1634/stemcells.2007-1009CrossRefPubMedGoogle Scholar
  14. 14.
    Zhao X, Liu Z, Yu L, Zhang Y, Baxter P, Voicu H, Gurusiddappa S, Luan J, Su JM, Leung HC, Li XN (2012) Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma. Neuro-Oncology 14(5):574–583.  https://doi.org/10.1093/neuonc/nos061CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    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(5):391–403.  https://doi.org/10.1016/j.ccr.2006.03.030CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Clavreul A, Jean I, Preisser L, Chassevent A, Sapin A, Michalak S, Menei P (2009) Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy. In Vitro Cell Dev Biol Anim 45(9):500–511.  https://doi.org/10.1007/s11626-009-9215-4CrossRefPubMedGoogle Scholar
  17. 17.
    Sanden E, Eberstal S, Visse E, Siesjo P, Darabi A (2015) A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumors to be utilized for therapeutic assays. Sci Rep 5:12218.  https://doi.org/10.1038/srep12218CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hovestadt V, Jones DT, Picelli S, Wang W, Kool M, Northcott PA, Sultan M, Stachurski K, Ryzhova M, Warnatz HJ, Ralser M, Brun S, Bunt J, Jager N, Kleinheinz K, Erkek S, Weber UD, Bartholomae CC, von Kalle C, Lawerenz C, Eils J, Koster J, Versteeg R, Milde T, Witt O, Schmidt S, Wolf S, Pietsch T, Rutkowski S, Scheurlen W, Taylor MD, Brors B, Felsberg J, Reifenberger G, Borkhardt A, Lehrach H, Wechsler-Reya RJ, Eils R, Yaspo ML, Landgraf P, Korshunov A, Zapatka M, Radlwimmer B, Pfister SM, Lichter P (2014) Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing. Nature 510(7506):537–541.  https://doi.org/10.1038/nature13268CrossRefPubMedGoogle Scholar
  19. 19.
    Schwalbe EC, Williamson D, Lindsey JC, Hamilton D, Ryan SL, Megahed H, Garami M, Hauser P, Dembowska-Baginska B, Perek D, Northcott PA, Taylor MD, Taylor RE, Ellison DW, Bailey S, Clifford SC (2013) DNA methylation profiling of medulloblastoma allows robust subclassification and improved outcome prediction using formalin-fixed biopsies. Acta Neuropathol 125(3):359–371.  https://doi.org/10.1007/s00401-012-1077-2CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Capper D, Jones DTW, Sill M, Hovestadt V, Schrimpf D, Sturm D, Koelsche C, Sahm F, Chavez L, Reuss DE, Kratz A, Wefers AK, Huang K, Pajtler KW, Schweizer L, Stichel D, Olar A, Engel NW, Lindenberg K, Harter PN, Braczynski AK, Plate KH, Dohmen H, Garvalov BK, Coras R, Hölsken A, Hewer E, Bewerunge-Hudler M, Schick M, Fischer R, Beschorner R, Schittenhelm J, Staszewski O, Wani K, Varlet P, Pages M, Temming P, Lohmann D, Selt F, Witt H, Milde T, Witt O, Aronica E, Giangaspero F, Rushing E, Scheurlen W, Geisenberger C, Rodriguez FJ, Becker A, Preusser M, Haberler C, Bjerkvig R, Cryan J, Farrell M, Deckert M, Hench J, Frank S, Serrano J, Kannan K, Tsirigos A, Brück W, Hofer S, Brehmer S, Seiz-Rosenhagen M, Hänggi D, Hans V, Rozsnoki S, Hansford JR, Kohlhof P, Kristensen BW, Lechner M, Lopes B, Mawrin C, Ketter R, Kulozik A, Khatib Z, Heppner F, Koch A, Jouvet A, Keohane C, Mühleisen H, Mueller W, Pohl U, Prinz M, Benner A, Zapatka M, Gottardo NG, Driever PH, Kramm CM, Müller HL, Rutkowski S, von Hoff K, Frühwald MC, Gnekow A, Fleischhack G, Tippelt S, Calaminus G, Monoranu C-M, Perry A, Jones C, Jacques TS, Radlwimmer B, Gessi M, Pietsch T, Schramm J, Schackert G, Westphal M, Reifenberger G, Wesseling P, Weller M, Collins VP, Blümcke I, Bendszus M, Debus J, Huang A, Jabado N, Northcott PA, Paulus W, Gajjar A, Robinson GW, Taylor MD, Jaunmuktane Z, Ryzhova M, Platten M, Unterberg A, Wick W, Karajannis MA, Mittelbronn M, Acker T, Hartmann C, Aldape K, Schüller U, Buslei R, Lichter P, Kool M, Herold-Mende C, Ellison DW, Hasselblatt M, Snuderl M, Brandner S, Korshunov A, von Deimling A, Pfister SM (2018) DNA methylation-based classification of central nervous system tumours. Nature 555(7697):469–474CrossRefGoogle Scholar
  21. 21.
    Boulay G, Awad ME, Riggi N, Archer TC, Iyer S, Boonseng WE, Rossetti NE, Naigles B, Rengarajan S, Volorio A, Kim JC, Mesirov JP, Tamayo P, Pomeroy SL, Aryee MJ, Rivera MN (2017) OTX2 activity at distal regulatory elements shapes the chromatin landscape of group 3 medulloblastoma. Cancer Discov 7(3):288–301.  https://doi.org/10.1158/2159-8290.CD-16-0844CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Lee Y, Miller HL, Jensen P, Hernan R, Connelly M, Wetmore C, Zindy F, Roussel MF, Curran T, Gilbertson RJ, McKinnon PJ (2003) A molecular fingerprint for medulloblastoma. Cancer Res 63(17):5428–5437PubMedGoogle Scholar
  23. 23.
    Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052):1707–1710CrossRefGoogle Scholar
  24. 24.
    Reynolds BA, Rietze RL (2005) Neural stem cells and neurospheres—re-evaluating the relationship. Nat Methods 2(5):333–336.  https://doi.org/10.1038/nmeth758CrossRefPubMedGoogle Scholar
  25. 25.
    Galli R (2013) The neurosphere assay applied to neural stem cells and cancer stem cells. Methods Mol Biol 986:267–277.  https://doi.org/10.1007/978-1-62703-311-4_17CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sara Badodi
    • 1
  • Silvia Marino
    • 1
    Email author
  • Loredana Guglielmi
    • 1
  1. 1.Blizard Institute, Barts and The London School of Medicine and DentistryQueen Mary University of LondonLondonUK

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