Abstract
Tumor resistance to conventional therapies is a major challenge toward the eradication of cancer, a life-threatening disease. This resistance mainly results from tumor heterogeneity and more specifically from the existence of “stem-like” cells that remain in a quiescent state for long periods of time and thus escape commonly used anti-cancer drugs resulting in treatment failure. Therefore, targeting this subpopulation would present a viable strategy to overcome tumor burden. This daunting task requires a deep and thorough understanding of the biology of the quiescent stem-cell population, their interaction with tumor microenvironments, and mechanisms used to sustain themselves despite aggressive therapies. In this chapter, we describe detailed technical procedures for the isolation of quiescent or infrequently dividing stem-like cells in cultured glioblastoma tumor cells using carboxy fluorescein succinimidyl ester (CFSE) staining and flow cytometric analysis. Quiescent glioblastoma cells with stem-like features are characterized and subsequently isolated based on their ability to retain the CFSE labeling.
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Veliz I, Loo Y, Castillo O, Karachaliou N, Nigro O, Rosell R (2015) Advances and challenges in the molecular biology and treatment of glioblastoma-is there any hope for the future? Ann Transl Med 3(1):7. doi:10.3978/j.issn.2305-5839.2014.10.06
Mrugala MM (2013) Advances and challenges in the treatment of glioblastoma: a clinician’s perspective. Discov Med 15(83):221–230
Dick JE (2008) Stem cell concepts renew cancer research. Blood 112(13):4793–4807. doi:10.1182/blood-2008-08-077941
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(Pt 5):1331–1343. doi:10.1093/brain/awr081
Jackson M, Hassiotou F, Nowak A (2015) Glioblastoma stem-like cells: at the root of tumor recurrence and a therapeutic target. Carcinogenesis 36(2):177–185. doi:10.1093/carcin/bgu243
Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB (2004) Identification of human brain tumour initiating cells. Nature 432(7015):396–401. doi:10.1038/nature03128
Son MJ, Woolard K, Nam DH, Lee J, Fine HA (2009) SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4(5):440–452. doi:10.1016/j.stem.2009.03.003
Rasper M, Schafer A, Piontek G, Teufel J, Brockhoff G, Ringel F, Heindl S, Zimmer C, Schlegel J (2010) Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity. Neuro Oncol 12(10):1024–1033. doi:10.1093/neuonc/noq070
Bleau AM, Hambardzumyan D, Ozawa T, Fomchenko EI, Huse JT, Brennan CW, Holland EC (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 4(3):226–235. doi:10.1016/j.stem.2009.01.007
Krishnamurthy K, Wang G, Rokhfeld D, Bieberich E (2008) Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide. Breast Cancer Res 10(6):R106. doi:10.1186/bcr2211
Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T, Herlyn M (2010) A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell 141(4):583–594. doi:10.1016/j.cell.2010.04.020
Dembinski JL, Krauss S (2009) Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma. Clin Exp Metastasis 26(7):611–623. doi:10.1007/s10585-009-9260-0
Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L, Holyoake TL (2002) Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood 99(1):319–325
Lyons AB (2000) Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J Immunol Methods 243(1–2):147–154
Deleyrolle LP, Rohaus MR, Fortin JM, Reynolds BA, Azari H (2012) Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE). J Vis Exp 62. doi:10.3791/3918
Azari H, Millette S, Ansari S, Rahman M, Deleyrolle LP, Reynolds BA (2011) Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay. J Vis Exp 56:e3633. doi:10.3791/3633
Rahman M, Reyner K, Deleyrolle L, Millette S, Azari H, Day BW, Stringer BW, Boyd AW, Johns TG, Blot V, Duggal R, Reynolds BA (2015) Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines. Anat Cell Biol 48(1):25–35. doi:10.5115/acb.2015.48.1.25
Acknowledgments
This work was supported by the Florida Center for Brain Tumor Research, the Preston A. Wells Jr. Center for Brain Tumor Therapy, the National Institutes of Health and the American Cancer Society.
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Azari, H., Deleyrolle, L.P., Reynolds, B.A. (2018). Using Carboxy Fluorescein Succinimidyl Ester (CFSE) to Identify Quiescent Glioblastoma Stem-Like Cells. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_4
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DOI: https://doi.org/10.1007/978-1-4939-7371-2_4
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