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ARTICLE ABSTRACTPurpose: Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system with an often lethal outcome due to metastatic disease. Migration and epithelial–mesenchymal transitions have been implicated in metastasis but they are hardly investigated in neuroblastoma.Experimental Design: Cell migration of 16 neuroblastoma cell lines was quantified in Transwell migration assays. Gene expression profiling was used to derive a migration signature, which was applied to classify samples in a neuroblastoma tumor series. Differential expression of transcription factors was analyzed in the subsets. NOTCH3 was prioritized, and inducible transgene expression studies in cell lines were used to establish whether it functions as a master switch for motility.Results: We identified a 36-gene expression signature that predicts cell migration. This signature was used to analyse expression profiles of 88 neuroblastoma tumors and identified a group with distant metastases and a poor prognosis. This group also expressed a known mesenchymal gene signature established in glioblastoma. Neuroblastomas recognized by the motility and mesenchymal signatures strongly expressed genes of the NOTCH pathway. Inducible expression of a NOTCH intracellular (NOTCH3-IC) transgene conferred a highly motile phenotype to neuroblastoma cells. NOTCH3-IC strongly induced expression of motility- and mesenchymal marker genes. Many of these genes were significantly coexpressed with NOTCH3 in neuroblastoma, as well as colon, kidney, ovary, and breast tumor series.Conclusion: The NOTCH3 transcription factor is a master regulator of motility in neuroblastoma. A subset of neuroblastoma with high expression of NOTCH3 and its downstream-regulated genes has mesenchymal characteristics, increased incidence of metastases, and a poor prognosis. Clin Cancer Res; 19(13); 3485–94. ©2013 AACR.