Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Abstract

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies 1 . To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia 2-8 . The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma 2,7,8 . The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds 9 . The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division 10 . CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity 2 . This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry 2 . The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Dual MAPK Inhibition Triggers Pro-inflammatory Signals and Sensitizes BRAFV600EGlioma to T Cell-Mediated Checkpoint Therapy

“…Due to their increased ability for DNA repair [81,82], mesenchymal transition [112], metabolic adaptation [113], and slow and asymmetric proliferation modes [114][115][116], CSCs are thought to survive standard chemoradiation better than non-stem-like cells; they are therefore potential culprits for therapy resistance and recurrence in multiple human cancers [117][118][119], such as leukemia [120], pancreatic cancer [121], colon cancer [122], and brain tumors [123]. Recent reports suggest that CD133 is a prognostic marker for relapse [124,125], and independent risk factor for progression and marker for poor clinical outcome in glioma patients [126,127], further underscoring the clinical importance of this marker.…”

Current approaches in identification and isolation of cancer stem cells

“…Another fluorescent dye that has been used to track the cell division frequency in several types of solid tumors is carboxyfluorescein succinimidyl ester (CFSE). Current studies have found that CFSE labeling can be used for identifying and isolating slow proliferating population cells from glioblastomas (Deleyrolle et al, 2011;Deleyrolle, Rohaus, Fortin, Reynolds, & Azari, 2012). These dyeretaining brain tumor cell populations are enriched in CSCs, and the progenitors that differentiate from these label-retaining cells exhibit all the pathological properties of the primary disease (Duan et al, 2013).…”