Abstract A19: Transformation of primary neural crest cells to model pediatric cancers

Neural crest cells (NCCs) are a multipotent, highly migratory cell population specified from the neural tube during embryonic development. NCCs undergo EMT (epithelial to mesenchymal transition) then migrate throughout the body forming diverse lineages including neurons, Schwann cells, melanocytes, and osteoblasts. Precursor cells from NCC lineages are thought to be the cell of origin for several pediatric and adult cancers including neuroblastoma, peripheral primitive neuroectodermal tumors (pPNET), malignant peripheral nerve sheath tumors (MPNST), cranial-facial osteosarcoma, and melanoma. The most common pediatric cancer in infants is neuroblastoma, which arises from the sypathoadrenal lineage of trunk NCCs. To study early events in neuroblastoma oncogenesis we established a system based on transformation of primary mouse NCCs. Trunk NCCs were isolated from day E9.5 embryos, and resulted in a population that was >95% positive for the NCC markers Sox10, p75, and Ascl1 (MASH1) by immunofluorescence. Culturing primary NCCs in neurogenic differentiation media resulted in neurons positive for Tuj1, Map2, and tyrosine hydroxylase (TH). Since a prominent category of high-risk neuroblastoma involves amplification of MYCN, we next determined whether MYCN overexpression was sufficient to transform wild-type NCCs in our assay. NCCs were isolated from C57Black6 mice and infected with MYCN retrovirus within 48hr of isolation, and then 10,000 cells/mouse were injected subcutaneously. MYCN overexpression alone generated a single tumor in 1 out of 12 mice, and the resulting tumor expressed the neuronal markers synaptophysin and tyrosine hydroxylase (TH) by immunohistochemistry (IHC). To determine whether loss of p53 could increase the transformation efficiency, NCCs from p53-compromised mice were infected with MYCN. These tumors had increased penetrance with 19 out of 19 mice developing tumors whether cells were injected into nude mice or into syngeneic C57Black6 mice with an intact immune system. Based on pathological analysis, these tumors were classified as primitive neuroectodermal tumors with divergent differentiation. Tumor regions which were diffusely positive for neuronal markers appeared neuroblastoma-like or PNET-like. Osteosarcoma was also a prominent feature in tumors with loss of p53. We also observed an additional tumor type which was negative by IHC for neuronal, Schwannian, and melanoma markers, and is not yet fully characterized. In general, mice with