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][3][4][5][6][7][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. Video LinkThe Gliomasphere culture is established and maintained using the neurosphere assay (NSA) as previously described 2,11,12 . 2. At the appropriate time for passaging the gliomaspheres, the medium containing the spheres is removed, placed in an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm (110 g) for 5 min at room temperature. 3. The supernatant is discarded and the pellet of spheres is resuspended in 1 ml of 0.05% trypsin-EDTA and incubated at 37°C in a water bath for 3-5 min. 4. An equal volume of soybean trypsin inhibitor is then used to stop the trypsin activity. 5. The cell suspension is gently pipetted up and down to ensure homogeneity and complete neutralization of the trypsin activity. 6. The cell suspension is again centrifuged at 800 rpm for 5 min. The supernatant is removed and the cells are resuspended in 1ml of NeuroCult NSC Basal Medium. 7. 10μl of the single-cell s...

Chromatin Remodeling Factor Brg1 Supports the Early Maintenance and Late Responsiveness of Nestin-Lineage Adult Neural Stem and Progenitor Cells

“…Following the last passage, neurospheres were labeled with carboxyfluorescein succinimidyl ester (CFSE) dye and treated with 4OH‐Tam. Dissociated cells were incubated with anti‐CD15 antibody for fluorescence‐activated cell sorting (FACS) .…”

Strategies for Isolating and Enriching Cancer Stem Cells: Well Begun Is Half Done

“…Recent studies have found that CFSE labeling can be used to identify and isolate a slow proliferating population of cells from glioblastomas. These dye-retaining brain tumor cell populations are enriched in CSCs, and the progeny that differentiate from these labelretaining cells exhibit all the pathological features of the primary disease [164,165]. Kamohara et al used Hoechst 33342 and Pyronin Y to sort Huh7 cells into G0, G1, and G2/M fractions by FACS.…”

NFATC4 Promotes Quiescence and Chemotherapy Resistance in Ovarian Cancer

“…Due to the low sensitivity of HEY1 to cisplatin at baseline, animals were treated with 2 daily doses of high-dose paclitaxel (16 mg/kg). While control tumors demonstrate a complete response to chemotherapy, tumors in which IcNFATC4 expression was transiently induced demonstrated growth arrest in response to doxycycline, and then ~8 days after 13 doxycycline discontinuation, tumors resumed normal growth without any evidence of response to therapy (p<0.001) ( Fig. 6e).…”

“…To agnostically determine if NFATC4 was enriched in slower proliferating cells, we used evaluated NFATC4 expression in slowly proliferating/vital dye retaining cells 13 in multiple ovarian cancer cell lines. Slow growing/dye retaining cells (Bright), demonstrated a significant enrichment for NFATC4 compared to the fast-growing cells (dim) in all four cell lines tested (HEY1 p<0.05, OVSAHO p<0.001, CaOV3 p<0.01, COV362 p<0.05) ( Fig.…”