“Side Population” Cells in Mouse Neuroblastoma Express Abcg2 and Are Enriched for Neuroblastoma Stem Cells.

There is now accumulating evidence for the existence of rare cancer stem cells that resemble adult stem cells in their ability to replicate and produce more specialized cells constituting the bulk of the tumor. Neuroblastoma is the most common childhood cancer, developing extracranially from neuroblasts of the body. Approximately 70–80% of patients have metastatic disease at the time of diagnosis, and fewer than half of these patients are cured. Human neuroblastoma cells have been shown to contain a subpopulation of cells with a high capacity to efflux Hoechst 33342 nuclear dye, resulting in a distinct side population (SP) phenotype (Hirschmann-Jax et al., PNAS, 2004). These SP cells also express high levels of ABCG2 and ABCA3 transporter genes. We have used a mouse model to further investigate the relationship between the SP phenotype and Abcg2 expression in neuroblastoma stem cells. Mice, expressing N-myc in neural-crest cells, develop neuroblastomas at early age (Weiss et al., EMBO J, 1997). We have found that these neuroblastomas can be divided into three groups according to their SP phenotype;

1. no SP cells present,

2. low SP cells (0.6–2% of total cell number) and

3. high SP cells (20–40% SP cells in total neuroblastoma cell population).

When present, the SP fraction was significantly decreased after treatment of the cells with gleevec and fumitremorgin C, inhibitors of Abcg2 function ( 4.7% with treatment vs. 30.5% untreated in one case). This result indicates that Abcg2 is a major determinant of SP phenotype in these tumors. Quantitative PCR, performed on sorted SP and non-SP cells confirmed about 6 fold higher level of Abcg2 expression in the SP cell fraction in comparison with non-SP. In order to determine the clonogenic capacity of different tumor cell populations, varying numbers of tumor cells were injected in the flanks of NOD/SCID/gamma null mice. Transplantability of the tumors was found to correlate with SP phenotype. At a dose of 106 cells per recipient, neuroblastomas with no SP cells did not form tumors (0 tumors developed in 6 recipients). Neuroblastoma cells with low SP cell numbers (0.6% of total cells) formed tumors in 2 out of 4 transplants at this cell dose. Neuroblastomas with a high SP cell population (30% of the cells) had the highest clonogenic activity, forming tumors in 6 out of 6 transplants at 106 cells per injection. These results indicate that tumor stem cells are more abundant in high SP tumors in comparison with tumors with lower SP cell fractions. Next, sorting experiments based on the SP phenotype indicated that SP cells are enriched for neuroblastoma stem cells. In one experiment using a neuroblastoma sample with 22% SP cells, recipients were inoculated with a dose of 105 sorted cells. Three out of 4 mice formed tumors after transplantation with sorted SP cells while only 1 of 4 mice transplanted with non-SP cells developed tumors. Secondary tumors, developed from sorted SP cells, had themselves higher proportion of SP cells in comparison with tumors, developed from non-SP cells (35–40% and 8–10%, respectively). We are now using this genetic mouse model to further study the use of Abcg2 expression to isolate neuroblastoma stem cells.