Advertisement

A Cell Culture System That Mimics Chronic Lymphocytic Leukemia Cells Microenvironment for Drug Screening and Characterization

  • Alessandro Natoni
  • Michael O’Dwyer
  • Corrado Santocanale
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 986)

Abstract

Chronic Lymphocytic Leukaemia (CLL) is an incurable disease that warrants new therapeutic treatments. CLL cells accumulate in the peripheral blood, in the bone marrow and in secondary lymphoid organs. Unlike circulating CLL cells, CLL cells resident in these last two compartments display high chemoresistance and proliferative capacity. Given the importance of the microenvironment in this disease, strategies that aim to develop new therapeutic agents need to consider this critical factor. Various cell culture conditions have been described that attempt to emulate either the different types of microenvironments in which CLL cells are found or an individual component of a particular microenvironment. Here, a methodology that partially mimics the interaction between CLL cells and the CD3+ CD4+ CD154+ T cells is described. Moreover, within this method, two protocols are presented and compared that may partially recapitulate different physiological states. The methodology can be exploited for target validation and drug development in CLL.

Key words

CLL Microenvironment T cells Chemotherapy Lymph Nodes Cell Death 

References

  1. 1.
    Lanasa MC (2010) Novel insights into the biology of CLL. Hematology Am Soc Hematol Educ Program 2010:70–76PubMedCrossRefGoogle Scholar
  2. 2.
    Burger JA (2011) Nurture versus nature: the microenvironment in chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program 2011:96–103PubMedCrossRefGoogle Scholar
  3. 3.
    Ghia P, Chiorazzi N, Stamatopoulos K (2008) Microenvironmental influences in chronic lymphocytic leukaemia: the role of antigen stimulation. J Intern Med 264:549–562PubMedCrossRefGoogle Scholar
  4. 4.
    Herishanu Y, Perez-Galan P, Liu D et al (2011) The lymph node microenvironment promotes B-cell receptor signaling. NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117:563–574PubMedCrossRefGoogle Scholar
  5. 5.
    Jaksic O, Kardum-Skelin I, Jaksic B (2010) Chronic lymphocytic leukemia: insights from lymph nodes & bone marrow and clinical perspectives. Coll Antropol 34:309–313PubMedGoogle Scholar
  6. 6.
    Chiorazzi N (2007) Cell proliferation and death: forgotten features of chronic lymphocytic leukemia B cells. Best Pract Res Clin Haematol 20:399–413PubMedCrossRefGoogle Scholar
  7. 7.
    Granziero L, Ghia P, Circosta P et al (2001) Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 97:2777–2783PubMedCrossRefGoogle Scholar
  8. 8.
    Lampert IA, Wotherspoon A, Van Noorden S et al (1999) High expression of CD23 in the proliferation centers of chronic lymphocytic leukemia in lymph nodes and spleen. Hum Pathol 30:648–654PubMedCrossRefGoogle Scholar
  9. 9.
    Vandewoestyne ML, Pede VC, Lambein KY et al (2011) Laser microdissection for the assessment of the clonal relationship between chronic lymphocytic leukemia/small lymphocytic lymphoma and proliferating B cells within lymph node pseudofollicles. Leukemia 25:883–888PubMedCrossRefGoogle Scholar
  10. 10.
    Munk Pedersen I, Reed J (2004) Microenvironmental interactions and survival of CLL B-cells. Leuk Lymphoma 45:2365–2372PubMedCrossRefGoogle Scholar
  11. 11.
    Smit LA, Hallaert DY, Spijker R et al (2007) Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood 109:1660–1668PubMedCrossRefGoogle Scholar
  12. 12.
    Vogler M, Butterworth M, Majid A et al (2009) Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 113:4403–4413PubMedCrossRefGoogle Scholar
  13. 13.
    Buggins AG, Pepper CJ (2010) The role of Bcl-2 family proteins in chronic lymphocytic leukaemia. Leuk Res 34:837–842PubMedCrossRefGoogle Scholar
  14. 14.
    Natoni A, Murillo LS, Kliszczak AE et al (2011) Mechanisms of action of a dual Cdc7/Cdk9 kinase inhibitor against quiescent and proliferating CLL cells. Mol Cancer Ther 10:1624–1634PubMedCrossRefGoogle Scholar
  15. 15.
    Burger JA, Ghia P, Rosenwald A et al (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 114:3367–3375PubMedCrossRefGoogle Scholar
  16. 16.
    Hayden RE, Pratt G, Roberts C et al (2011) Treatment of chronic lymphocytic leukemia requires targeting of the protective lymph node environment with novel therapeutic approaches. Leuk Lymphoma 53(4):537–549PubMedCrossRefGoogle Scholar
  17. 17.
    Willimott S, Baou M, Huf S et al (2007) Separate cell culture conditions to promote proliferation or quiescent cell survival in chronic lymphocytic leukemia. Leuk Lymphoma 48:1647–1650PubMedCrossRefGoogle Scholar
  18. 18.
    Willimott S, Baou M, Naresh K et al (2007) CD154 induces a switch in pro-survival Bcl-2 family members in chronic lymphocytic leukaemia. Br J Haematol 138:721–732PubMedCrossRefGoogle Scholar
  19. 19.
    Neron S, Nadeau PJ, Darveau A et al (2011) Tuning of CD40-CD154 interactions in human B-lymphocyte activation: a broad array of in vitro models for a complex in vivo situation. Arch Immunol Ther Exp (Warsz) 59:25–40CrossRefGoogle Scholar
  20. 20.
    Pepper C, Mahdi JG, Buggins AG et al (2011) Two novel aspirin analogues show selective cytotoxicity in primary chronic lymphocytic leukaemia cells that is associated with dual inhibition of Rel A and COX-2. Cell Prolif 44:380–390PubMedCrossRefGoogle Scholar
  21. 21.
    Hayden RE, Pratt G, Davies NJ et al (2009) Treatment of primary CLL cells with bezafibrate and medroxyprogesterone acetate induces apoptosis and represses the pro-proliferative signal of CD40-ligand, in part through increased 15dDelta12,14, PGJ2. Leukemia 23:292–304PubMedCrossRefGoogle Scholar
  22. 22.
    Jak M, van Bochove GG, van Lier RA et al (2011) CD40 stimulation sensitizes CLL cells to rituximab-induced cell death. Leukemia 25:968–978PubMedCrossRefGoogle Scholar
  23. 23.
    McCaig AM, Cosimo E, Leach MT et al (2011) Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals. Br J Haematol 153(2):199–211PubMedCrossRefGoogle Scholar
  24. 24.
    Tromp JM, Geest CR, Breij EC et al (2012) Tipping the Noxa/Mcl-1 balance overcomes ABT-737 resistance in chronic lymphocytic leukemia. Clin Cancer Res 18:487–498PubMedCrossRefGoogle Scholar
  25. 25.
    Jak M, van Bochove GG, Reits EA et al (2011) CD40 stimulation sensitizes CLL cells to lysosomal cell death induction by type II anti-CD20 mAb GA101. Blood 118:5178–5188PubMedCrossRefGoogle Scholar
  26. 26.
    Tonino SH, van Laar J, van Oers MH et al (2011) ROS-mediated upregulation of Noxa overcomes chemoresistance in chronic lymphocytic leukemia. Oncogene 30:701–713PubMedCrossRefGoogle Scholar
  27. 27.
    Dietrich S, Kramer OH, Hahn E et al (2012) Leflunomide induces apoptosis in fludarabine-resistant and clinically refractory CLL cells. Clin Cancer Res 18:417–431PubMedCrossRefGoogle Scholar
  28. 28.
    Hayden RE, Pratt G, Drayson MT et al (2010) Lycorine sensitizes CD40 ligand-protected chronic lymphocytic leukemia cells to bezafibrate- and medroxyprogesterone acetate-induced apoptosis but dasatanib does not overcome reported CD40-mediated drug resistance. Haematologica 95:1889–1896PubMedCrossRefGoogle Scholar
  29. 29.
    Hallaert DY, Jaspers A, van Noesel CJ et al (2008) c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches. Blood 112:5141–5149PubMedCrossRefGoogle Scholar
  30. 30.
    Logue SE, Elgendy M, Martin SJ (2009) Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nat Protoc 4:1383–1395PubMedCrossRefGoogle Scholar
  31. 31.
    Baou M, Kohlhaas SL, Butterworth M et al (2010) Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells. Haematologica 95:1510–1518PubMedCrossRefGoogle Scholar
  32. 32.
    Wright SJ, Robertson LE, O’Brien S et al (1994) The role of fludarabine in hematological malignancies. Blood Rev 8:125–134PubMedCrossRefGoogle Scholar
  33. 33.
    Montagnoli A, Valsasina B, Croci V et al (2008) A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nat Chem Biol 4:357–365PubMedCrossRefGoogle Scholar
  34. 34.
    World Medical Organization (1996) Declaration of Helsinki. Br Med J 313:1448–1449Google Scholar

Copyright information

© SpringerScience+Business Media New York 2013

Authors and Affiliations

  • Alessandro Natoni
    • 1
  • Michael O’Dwyer
    • 2
  • Corrado Santocanale
    • 1
  1. 1.Centre for Chromosome Biology and National Centre of Biomedical Engineering and Science, School of Natural SciencesNational University of Ireland GalwayGalwayIreland
  2. 2.National Centre of Biomedical Engineering and Science, School of MedicineNational University of Ireland GalwayGalwayIreland

Personalised recommendations