Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. Based on the degree of neuronal differentiation, NB is histopathologically divided into undifferentiated (UD), poorly differentiated (PD) and differentiating subtypes. Recently, a unique histological subset of NBs within the UD and PD subtypes has been identified. These tumors are uniformly composed of large cells having vesicular nuclei with sharply outlined nuclear membranes, and one to four prominent nucleoli, which are referred to as “Large-Cell Neuroblastomas” or LCNs. Patients with the UD neuroblastoma with the LCN appearance had a very poor prognosis regardless of age at diagnosis, clinical stage, and DNA index. Surprisingly, non-MYCN amplified UDs behaved significantly worse than MYCN amplified UDs. Our hypothesis is that the histological features and the clinical behavior of LCNs are the reminiscence of a malignancy likely driven by stem cell-like cancer cells. To gain better understanding of the nature of LCNs, we have generated phenotypically stablilized stem cell-like NB cells by treating monolayer NB cell lines with epigenetic modifiers for a short time. Without further exposure to epigenetic modifiers, stemness phenotypes of the NB cells were maintained over a year in sphere-forming culture conditions. Hence, we referred to these spheres as induced CSCs (iCSCs). We previously presented that the SKNAS iCSC had a very high tumor-initiating ability in SCID/Beige mice and SKNAS iCSC xenografts recapitulated the histological appearance of totally undifferentiated “large-cell” NBs. A cancer stem cell marker, CXCR4, was preferentially expressed in the iCSC xenografts over the monolayer counterparts. In this study, we established additional iCSCs from three NB cell lines [SKNBE(2)C, CHP134, SY5Y]. The expression of stemness factors was augmented in these NB iCSCs compared to conventional monolayer counterparts. In addition, the expression of stem cell markers (CD24, CD133) and neural crest stem cell markers (SOX9, SLUG, Musashi, p75NTR) was elevated in these NB iCSCs. Notably, SCID/Beige mice subcutaneously injected with one hundred SKNBE(2)C iCSCs formed tumors, and in some cases, SKNBE(2)C iCSCs metastasized to adrenal gland, liver, spleen, kidney, indicating an increased tumor invasiveness of these cells. CXCR4 and p75NTR expression was positive in the iCSC xenografts, although not uniformly. By histopathological and immunohistochemical examinations, we confirmed that the xenografts established from SKNAS iCSC, SKNBE(2)C iCSC, CHP134 iCSC, and SY5Y iCSC shared two common and consistent features: the LCN phenotype and high-level expression of MYC/MYCN. These characteristics may help identify the presence of CSC compartment in patients’ specimens. The established NB iCSCs may also serve as useful tools for the development of effective therapeutics against NB.

Citation Format: Naohiko Ikegaki, Hiroyuki Shimada, Xao X. Tang. Induced stable neuroblastoma stem cells recapitulate in vivo highly aggressive large-cell neuroblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2654. doi:10.1158/1538-7445.AM2013-2654