B. T. Craig1, E. J. Rellinger1, A. L. Alvarez1, J. Qiao1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA
Introduction: Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, the mechanisms that are responsible for sphere formation remain poorly understood. We hypothesize that a dedifferentiation program mediates neuroblastoma sphere formation and that more stem cell-like cell lines will form spheres at a higher rate than their more differentiated counterparts.
Methods: Four human neuroblastoma cell lines (BE(2)-C, I-type, MYCN amplified; IMR-32, N-type, MYCN amplified; SK-N-SH, S-type, MYCN single copy; SK-N-AS, S-type, MYCN single copy) were examined for intrinsic frequency of sphere formation by limiting dilution analysis in serum-free media supplemented with EGF (20 ng/ml) and bFGF (40 ng/ml). 13-cis-retinoic acid (RA, 5 µM) is clinically used as a differentiating agent for high-risk neuroblastoma. Protein levels were assessed by SDS-PAGE. Multiple group comparisons were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Two group comparisons were analyzed by two-tailed unpaired Student’s t test. In all cases a p value of <0.05 was considered significant.
Results: Both MYCN amplified and MYCN single copy cell lines formed spheres, contrary to previous reports that this phenotype depended on MYCN amplification. BE(2)-C cells demonstrated the highest frequency of sphere formation and significantly differed from the other 3 cell lines (6.58% vs. 0.7%, 1.5% and 2.26%, p<0.05). bFGF regulated sphere formation in a dose-dependent fashion while EGF did not (20-60 ng/ml, 3-5x fold change, p<0.05). Serial passage in sphere culture conditions increased frequency of sphere formation two fold (p <0.05), and increased expression of the stem cell factor Oct4. The related factor Nanog did not change expression from baseline. 13-cis-RA (5 µM) nearly completely blocked sphere formation in the BE(2)-C cell line (6.58% vs. 0.57%, p<0.05).
Conclusion: BE(2)-C cells are I-type neuroblastoma cells that have previously been shown to exhibit stem cell features. BE(2)-C is the most avid sphere-forming cell line, and this phenotype is inhibited by induced differentiation with 13-cis-RA. Furthermore, sphere culture affected the expression level of a critical stem cell factor in a manner consistent with previous reports. Hence, the in vitro model system of sphere formation correlates with known parameters of stem cell-like behavior in neuroblastoma and is therefore a valid system for the study of neuroblastoma stem cells.