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EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1869)

Abstract

The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.

Key words

Mass cytometry (CyTOF) Synthetic antibodies Cancer stem cells Brain tumor initiating cells (BTICs) Glioblastoma and Eph receptors 
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References

  1. 1.
    Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111CrossRefGoogle Scholar
  2. 2.
    Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB (2009) Tumor-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8(10):806–823CrossRefGoogle Scholar
  3. 3.
    Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A (2007) Tumor growth need not be driven by rare cancer stem cells. Science 317(5836):337CrossRefGoogle Scholar
  4. 4.
    Yoo MH, Hatfield DL (2008) The cancer stem cell theory: is it correct? Mol Cells 26(5):514PubMedPubMedCentralGoogle Scholar
  5. 5.
    Rahman M, Deleyrolle L, Vedam-Mai V, Azari H, Abd-El-Barr M, Reynolds BA (2011) The cancer stem cell hypothesis: failures and pitfalls. Neurosurgery 68(2):531–545CrossRefGoogle Scholar
  6. 6.
    Bao B, Ahmad A, Azmi AS, Ali S, Sarkar FH (2013) Overview of cancer stem cells (CSCs) and mechanisms of their regulation: implications for cancer therapy. Curr Protoc Pharmacol:14–25Google Scholar
  7. 7.
    Neuzil J, Stantic M, Zobalova R, Chladova J, Wang X, Prochazka L, Dong L, Andera L, Ralph SJ (2007) Tumour-initiating cells vs. cancer ‘stem’cells and CD133: what’s in the name? Biochem Biophys Res Commun 355(4):855–859CrossRefGoogle Scholar
  8. 8.
    Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old idea—a paradigm shift. Cancer Res 66(4):1883–1890CrossRefGoogle Scholar
  9. 9.
    Gupta PB, Chaffer CL, Weinberg RA (2009) Cancer stem cells: mirage or reality? Nat Med 15(9):1010–1012CrossRefGoogle Scholar
  10. 10.
    Singh SK, Hawkins C, Clarke ID et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401.  https://doi.org/10.1038/nature03128CrossRefGoogle Scholar
  11. 11.
    Chen J, Li Y, Yu TS et al (2012) A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488:522–526.  https://doi.org/10.1038/nature11287CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Liu G, Yuan X, Zeng Z et al (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:67.  https://doi.org/10.1186/1476-4598-5-67CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mao X, Zhang X, Xue X et al (2009) Brain tumor stem-like cells identified by neural stem cell marker CD15. Transl Oncol 2(4):247–257CrossRefGoogle Scholar
  14. 14.
    Abdouh M, Facchino S, Chatoo W et al (2009) BMI1 sustains human glioblastoma multiforme stem cell renewal. J Neurosci 29(28):8884–8896CrossRefGoogle Scholar
  15. 15.
    Binda E, Visioli A, Giani F et al (2012) The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-propagating cells from human glioblastomas. Cancer Cell 22:765–780.  https://doi.org/10.1016/j.ccr.2012.11.005CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Day BW, Stringer BW, Al-Ejeh F et al (2013) EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme. Cancer Cell 23:238–248.  https://doi.org/10.1016/j.ccr.2013.01.007CrossRefPubMedGoogle Scholar
  17. 17.
    Day BW, Stringer BW, Boyd AW (2014) Eph receptors as therapeutic targets in glioblastoma. Br J Cancer 111:1255–1261.  https://doi.org/10.1038/bjc.2014.73CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10(3):165–180CrossRefGoogle Scholar
  19. 19.
    Lu-Emerson C, Norden AD, Drappatz J, Quant EC, Beroukhim R, Ciampa AS, Doherty LM, Lafrankie DC, Ruland S, Wen PY (2011) Retrospective study of dasatinib for recurrent glioblastoma after bevacizumab failure. J Neuro-Oncol 104(1):287–291CrossRefGoogle Scholar
  20. 20.
    Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK (2012) A deep profiler’s guide to cytometry. Trends Immunol 33(7):323–332CrossRefGoogle Scholar
  21. 21.
    Bandura DR, Baranov VI, Ornatsky OI et al (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81(16):6813–6822CrossRefGoogle Scholar
  22. 22.
    Bendall SC, Simonds EF, Qiu P et al (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030):687–696CrossRefGoogle Scholar
  23. 23.
    Fellouse F, Esaki K, Birtalan S et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373(4):924–940CrossRefGoogle Scholar
  24. 24.
    Miersch S, Sidhu SS (2012) Synthetic antibodies: concepts, potential and practical considerations. Methods 57(4):486–498CrossRefGoogle Scholar
  25. 25.
    Amir EAD, Davis KL, Tadmor MD et al (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol 31(6):545–552CrossRefGoogle Scholar
  26. 26.
    Lathia JD, Gallagher J, Heddleston JM, Wang J, Eyler CE, MacSwords J, Wu Q, Vasanji A, McLendon RE, Hjelmeland AB, Rich JN (2010) Integrin alpha 6 regulates glioblastoma stem cells. Cell Stem Cell 6(5):421–432CrossRefGoogle Scholar
  27. 27.
    Gangemi RM, Griffero F, Marubbi D, Perera M, Capra MC, Malatesta P, Ravetti GL, Zona GL, Daga A, Corte G (2009) SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity. Stem Cells 27(1):40–48CrossRefGoogle Scholar
  28. 28.
    Verginelli F, Perin A, Dali R et al (2013) Transcription factors FOXG1 and Groucho/TLE promote glioblastoma growth. Nat Commun 4Google Scholar
  29. 29.
    Günther HS, Schmidt NO, Phillips HS et al (2008) Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 27(20):2897–2909CrossRefGoogle Scholar
  30. 30.
    Qiu P, Simonds EF, Bendall SC et al (2011) Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat Biotechnol 29(10):886–891CrossRefGoogle Scholar
  31. 31.
  32. 32.
  33. 33.
    Finck R, Simonds EF, Jager A et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83(5):483–494CrossRefGoogle Scholar
  34. 34.

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

Authors and Affiliations

  1. 1.Department of Molecular GeneticsUniversity of TorontoTorontoCanada
  2. 2.Donnelly Centre for Cellular and Biomolecular ResearchUniversity of TorontoTorontoCanada
  3. 3.McMaster Stem Cell and Cancer Research InstituteMcMaster UniversityHamiltonCanada
  4. 4.Department of Biochemistry and Biomedical SciencesMcMaster UniversityHamiltonCanada
  5. 5.Department of SurgeryMcMaster UniversityHamiltonCanada
  6. 6.Canadian Institute for Advanced ResearchTorontoCanada
  7. 7.Stem Cell and Cancer Research InstituteMcMaster UniversityHamiltonCanada

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