Figure 2.

N-Myc transforms p53 compromised NCCs into metastatic primitive neuroectodermal tumors with divergent differentiation. (a) Approximately 10,000 p53^mixed or p53^+/− NCCs infected with N-Myc virus³⁶ were mixed with 100 μl Matrigel (BD Biosciences, San Jose, CA, USA, 356237) and injected subcutaneously on the right flank of 6- to 8-week-old either Nu/Nu mice or C57Bl/6 mice (Charles River, Wilmington, MA, USA). Mice were injected on a rolling basis, to generate sufficient numbers of tumors for further analysis in a non-randomized, non-blinded manner. Eight Nu/Nu mice (left panel) and six C57Bl/6 mice (middle panel) were each flank injected with N-Myc transduced primary p53^mixed NCCs pooled from p53^+/− and p53^−/− embryos (middle panel). Four C57Bl/6 mice were flank injected with N-Myc transduced primary p53^+/− NCCs (right panel). Individual tumor volumes were measured over time as previously described.³⁶ (b) Formalin-fixed tissue samples were prepared as previously described.³⁶ Representative tumor histology (hematoxylin and eosin, H&E) is shown from a region positive for neuronal markers (upper left) and a region with osteosarcoma (upper right). Representative immunohistochemistry images show expression of neuronal markers. Antibodies used include TH (1:40, Vector, Burlingame, CA, USA, VP-T489), Synaptophysin (1:100, Spring Biosciences, Pleasanton, CA, USA, E2172), MAP2 (1:250, Millipore, Billerica, MA, USA, AB5622) and Phox2B (1:100, Abcam, ab183741). Scale bars=50 μM. (c) Representative histology (H&E) of lymph node, liver and lung metastasis with areas of metastasis highlighted in the insets. Scale bars=50 μM. This experiment was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. Mice were sacrificed using CO2 asphyxiation followed by cervical dislocation. This research was approved by the St. Jude Children’s Research Hospital IACUC.