Abstract LB-255: A phenotypic and molecular characterization of murine neuroblastoma stem-like cells

Abstract

Abstract Reports suggest the existence of cancer stem cells in solid tumors and it appears that tumor cells responsible for failure after successful initial therapy exhibit stem cell-like properties. However, tumor cells that undergo transition between epithelial and mesenchymal states may also acquire stem-like traits and are thought to be activated during cancer invasion and metastasis. It is unclear whether such cells are true stem cells or simply gain stem-like properties. We used mouse neuroblastoma to develop a model where stem-like cells were generated and studied in the context of tumorigenicity, proliferation, response to radiation and chemotherapy. Next, we generated complete protein profiles of neuroblastoma stem-like (tumorspheres) and non stem-like (committed) cells comparing them to normal murine subventricular zone (SVZ) stem cells attempting to further characterize the “stem-ness” of these tumor cells. Murine neuroblastoma and the Neuro2A cell line were harvested and cultured in regular media with FBS or NeuroCult complete media specific for enrichment of neural stem cells. Adult mouse SVZ was dissected, and neural stem cells isolated and cultured in NeuroCult complete media. We describe a subset of mouse neuroblasoma cells harvested from established tumors and cell lines with characteristics of neural stem cells. This subset shows features of self-renewal, grows in tumorspheres in serum free media and can be stimulated to undergo multi-lineage differentiation. Moreover, stem-like cells showed higher resistance to radiation, doxorubicin and Sutent treatment compared to committed cells. Committed cells showed a higher rate of proliferation both in vitro and in vivo. Using clonogenic assays, there was cross over between stem-like and non-stem like cells whereby tumorspheres reseeded into full-serum media exhibited a fully-differentiated, epithelial-like phenotype. Likewise, fully-differentiated non-stem like cells, if reseeded into stem cell media, resulted in round, undifferentiated, non-adherent cells resembling tumorspheres. Stringent proteomic filtration identified 1,168 proteins. The greatest proteomic similarity was between stem-like and non stem-like cells with little overlap between SVZ stem cells (n=45 or n=51 respectively). Ninety two proteins were uniquely expressed by SVZ cells, including Nestin, defining a differential proteomic profile for neural stem cells compared to tumor stemlike cells. Several mesenchymal proteins were differentially up-regulated in the stem-like tumorspheres suggesting a mesenchymal transition. Our data show that neuroblastoma tumor cells can transition between the epithelial and mesenchymal state and such cells are not true stem cells. The mesenchymal cells have stem-like qualities, but on proteomic analysis differ significantly from neural stem cells. Characterization of the proteome, not only validated the phenotypes, but may also identify targets for selective killing of resistant tumor-specific mesenchymal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-255.