Abstract
Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.
Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable – together with incubation in low oxygen – to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cipolleschi MG, Rovida E, Dello SP (2013) The Culture-Repopulating Ability assays and incubation in low oxygen: a simple way to test drugs on leukaemia stem or progenitor cells. Curr Pharm Des 19:5374–5383
Giuntoli S, Rovida E, Gozzini A et al (2007) Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells. Stem Cells 25:1119–1125
Cipolleschi MG, Dello SP, Olivotto M (1993) The role of hypoxia in the maintenance of hematopoietic stem cells. Blood 82:2031–2037
Schofield R (1978) The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 4:7–25
Ng KP, Manjeri A, Lee KL et al (2014) Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition. Blood 123:3316–3326
Giuntoli S, Rovida E, Barbetti V et al (2006) Hypoxia suppresses BCR/Abl and selects imatinib-insensitive progenitors within clonal CML populations. Leukemia 20:1291–1293
Giuntoli S, Tanturli M, Di Gesualdo F et al (2011) Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate. Haematologica 96:204–212
Tanturli M, Giuntoli S, Barbetti V et al (2011) Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia. PLoS One 6:e17008
Del Poggetto E, Tanturli M, Ben-Califa N et al (2015) Salarin C inhibits maintenance of hypoxia-selected Chronic Myeloid Leukemia progenitor cells. Cell Cycle 14:3146–3154
Sullivan R, Pare GC, Frederiksen LJ et al (2008) Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity. Mol Cancer Ther 7:1961–1973
Schnitzer SE, Schmid T, Zhou J et al (2006) Hypoxia and HIF-1alpha protect A549 cells from drug-induced apoptosis. Cell Death Differ 13:1611–1613
Acknowledgment
The authors thank Prof. Persio Dello Sbarba for his time spent in reviewing the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cheloni, G., Tanturli, M. (2016). The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells. In: Li, S., Zhang, H. (eds) Chronic Myeloid Leukemia. Methods in Molecular Biology, vol 1465. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4011-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-4011-0_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-4009-7
Online ISBN: 978-1-4939-4011-0
eBook Packages: Springer Protocols