Neuroblastoma 3D cell culture: Proteomic differences between cancer spheroids and monolayers

Abstract

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Solid tumors, such as neuroblastoma (NB), are associated with a heterogeneous cell environment. Multicellular tumor spheroid (MCTS) cultures have been shown to better mimic growth characteristics of in vivo solid tumors. Because tumor spheroid growth patterns may be quite different from standard two-dimensional culture systems, we compared the protein expression profiles of two- and three-dimensional in vitro NB cultures, ie, monolayers and MCTS. Human NB cells (SK-N-AS, SK-N-DZ, and IMR-32) were grown as both monolayers and spheres. Enriched nuclear/cytosolic protein fractions were analyzed for differentially secreted proteins by two- dimensional polyacrylamide gel electrophoresis (2-D PAGE). Selected polypeptides were identified by liquid chromatography linked tandem mass spectrometry (LC-MS/MS). Cell cycle division (septin 2), glycolysis (transketolase, triosephosphate isomerase, pyruvate kinase M1/M2, alpha enolase, and phosphoglycerate mutase-1), cell stress (HSPs 90, 70, and 60), structural (adenyl CAP-1, tubulin β-2, and cofilin), and signal transduction (peptidyl prolyl cis/trans isomerase A) polypeptides were overexpressed in spheroids. As many of these proteins have links to cancer, tumor cells established as 3-D NB spheroids as compared to traditional monolayers may have important physiological and functional significance. These cell specific proteins may be of physiological significance in 3-D NB spheroids as compared to traditional monolayer cell growth. The altered proteins among NB spheroids may represent an important link between monolayer cell cultures and in vivo experiments and thus a more ideal in vitro culture system for determining the precise three-dimensional microenvironment of NB.