Expression of Mycn in migrating mouse NCC leads to tumor formation in immunocompetent, syngeneic C57BL/6J hosts. (A) Neural tubes (NT) from E9.5 C57BL/6J embryos were dissected between somite 16 and 24 (indicated by red lines) and cultivated in vitro on laminin-coated plates for 24 h. The migrating NCC were transduced with a Mycn-IRES-GFP retroviral construct. GFP-positive cells were sorted and formed spheres. (B) Mycn-expressing NCC (green, n = 13) and NCC transduced with an empty vector (black, n = 10) were injected in the flank of C57BL/6J mice. Tumor formation was monitored over time until endpoint was reached. (C) H&E and IHC staining for N-Myc, Phox2B, TH, synaptophysin, and NCAM on Mycn-nGEMM tumor shows high expression of all markers. Scale bar = 50 µm. (D) Mycn expression relative to HPRT as assessed by qRT-PCR in Mycn-nGEMM-high and Mycn-nGEMM-low cell lines. Data represent mean values ± SD (n = 2 independent experiments). (E) N-Myc expression assessed by western blot in Mycn-nGEMM-high and Mycn-nGEMM-low tumors. (F) Growth curves for Mycn-nGEMM-high (n = 6 mice) and Mycn-nGEMM-low (n = 7 mice) tumors as assessed by BLI. Data represent mean values ± SEM. (G) Kaplan–Meier curves for C57BL/6J mice injected with Mycn-nGEMM-high or Mycn-nGEMM-low cell lines in the renal capsule (n = 5 mice per group). Kaplan–Meier survival analyses were performed by log-rank test. **P < 0.01 (H) t-SNE clustering including TH-MYCN, Mycn-nGEMM, NCC, and Mycn-NCC samples and indicated human cancer datasets. (I) UMAP plot using GMKF dataset depending on MYCN-amplification. Source data are available for this figure: SourceData F1.