Neural crest cells (NCCs) are the embryonic precursor cell population to neuroblastoma. To study neuroblastoma oncogenesis we have established an assay based on the oncogenic transformation of primary mouse NCCs. Since N-Myc amplification is a prominent category of high-risk neuroblastoma, we first determined if N-Myc overexpression was sufficient to transform NCCs. Primary NCCs were isolated from neural tube explants from day 9.5 embryos. We confirmed isolation of NCCs by immunostaining for NCC markers and their ability to form tyrosine hydroxylase (TH) positive neurons. NCCs were retrovirally transduced with MSCV-N-Myc and ~10,000 cells were injected subcutaneously per mouse resulting in two tumors out of 16 mice. Both tumors were uniformly positive for synaptophysin, MAP2 and Phox2B with sporadic staining for TH. These tumors contained neuropil and ganglion cells, and were characterized as neuroblastoma. To determine if loss of p53 could increase the efficiency of transformation, NCCs from p53-compromised mice were infected with N-Myc. This led to 100% tumor penetrance in 19 mice. These tumors were a mix of PNET and other cancer types, and were characterized as primitive neuroectodermal tumors with divergent differentiation. The NCC approach we have established is a promising new tool for investigating the molecular causes and contribution of impaired development to neuroblastoma.
Using our NCC system we can test treatment response along neuroblastoma ontogeny. Stem/progenitor cells, like NCCs, are believed to be the cell of origin for many cancers and a source for the cancer-initiating cells that are speculated to cause relapse. BET (bromodomain and extraterminal domain) inhibitors, like JQ1, target shared lineages in some cancers like SCLC. We too found a lineage-dependent sensitivity in neuroblastoma with superimposable JQ1 responses seen from non-transformed wild-type NCCs through NCC-derived neuroblastomas. We found no correlation of JQ1-sensitivity with proliferation rates, N-Myc expression, transformation status or p53 status. This suggests BET bromodomain inhibitors can target the immature cell populations of neuroblastoma and should be explored as a therapy for preventing relapse.