Quantification, self-renewal, and genetic tracing of FL1⁺ tumor-initiating cells in a large cohort of human gliomas

Abstract

Evidence has emerged that the initiation and growth of gliomas is sustained by a subpopulation of cancer-initiating cells (CICs). Because of the difficulty of using markers to tag CICs in gliomas, we have previously exploited more robust phenotypic characteristics, including a specific morphology and intrincic autofluorescence, to identify and isolate a subpopulation of glioma CICs, called FL1(+). The objective of this study was to further validate our method in a large cohort of human glioma and a mouse model of glioma. Seventy-four human gliomas of all grades and the GFAP-V(12)HA-ras B8 mouse model were analyzed for in vitro self-renewal capacity and their content of FL1(+). Nonneoplastic brain tissue and embryonic mouse brain were used as control. Genetic traceability along passages was assessed with microsatellite analysis. We found that FL1(+) cells from low-grade gliomas and from control nonneoplasic brain tissue show a lower level of autofluorescence and undergo a restricted number of cell divisions before dying in culture. In contrast, we found that FL1(+) cells derived from many but not all high-grade gliomas acquire high levels of autofluorescence and can be propagated in long-term cultures. Moreover, FL1(+) cells show a remarkable traceability over time in vitro and in vivo. Our results show that FL1(+) cells can be found in all specimens of a large cohort of human gliomas of different grades and in a model of genetically induced mouse glioma as well as nonneoplastic brain. However, their self-renewal capacity is variable and seems to be dependent on the tumor grade.

Fig. 1.

FL1⁺ cells are detected in gliomas and in (nonnepolastic) tissue samples resected from patients with chronic, nonlesional epilepsy. Left panel represents pictures of different regions within non-neoplastic brain resected from epilepsy patients and gliomas of grades I, II, III, and IV stained with H&E. Right panels are representative dot plot FACS of freshly dissociated tissue obtained through epilepsy surgery and gliomas of grades I, II, III, and IV. P1 gates viable FL1⁺ cells while P2 gates viable FL1⁰ cells.

Fig. 2.

Graphs show (A) the percentage of FL1⁺ cells in various glioma grades compared to nonneoplastic brain, (B) the percentage of long-term cell cultures in various tumor grades and epileptic tissue samples, (C) the intensity of autofluorescence detected at 515 ± 15 nm in the FL1⁺ cell cultures derived from various tumor grades and epileptic tissues.

Fig. 3.

Proportion of FL1⁺ cells in epileptic tissue (A) and gliomas (A–C) cultured in serum-free conditions (A–C) and in vivo over several passages (D). R1 gates viable FL1⁺ cells while R2 gates viable FL1⁰ cells.

Fig. 4.

FL1⁺ cells are detected in neonatal, in postnatal mouse brain tissues (A) and in mouse brain of symptomatic rasB8 mice (C). A represents a dot plot FACS of dissociated mouse brain at E11 (left panel), P0-1 (middle panel), and P21 (right panel). B and D represent the quantification of the percentage viable FL1⁺ cells at the different stages of mouse development and in mouse brain of symptomatic rasB8 mice, respectively. P1 gates viable FL1⁺ cells while P2 gates viable FL1⁰ cells.