Modeling Pediatric Malignancies By Transforming Primary Neural Crest Cells

Abstract Neural crest cells (NCCs) are a highly migratory multipotent cell population first specified during neural tube closure at the neural plate border. Soon after neural tube closure NCCs undergo epithelial to mesenchymal transitioning (EMT) to allow migration throughout the body plan where they generate diverse cell types including neurons, Schwann cells, melanocytes, and osteoblasts. NCCs have been proposed to be the embryonic precursor cell population to multiple pediatric malignancies including neuroblastoma, peripheral primitive neuroectodermal tumors (pPNET), malignant peripheral nerve sheath tumors (MPNST), cranial-facial osteosarcoma as well as adult malignancies like melanoma. Despite this very little work has been done with primary NCCs in pediatric cancer research. The most common of these pediatric cancers is neuroblastoma, which arises in the sympathoadrenal lineage of trunk NCCs. To establish a system for studying the earliest events in neuroblastoma oncogenesis we developed an assay based on the oncogenic transformation of primary NCCs. Isolation of primary trunk NCCs from day 9.5 embryos resulted in a population that was >95% positive for the NCC markers Sox10, p75 and Ascl1 (MASH1) as assessed by immunofluorescence. These isolated NCCs were also capable of generating Tuj1, Map2, and tyrosine hydroxylase (TH) positive neurons when placed in neurogenic media. We next wanted to test the transformation of primary NCCs. Since a prominent category of high-risk neuroblastoma is N-Myc amplification, we first determined if N-Myc overexpression was sufficient to transform wild-type NCCs in our assay. For this and all other tumor experiments, NCCs were retrovirally infected within 48 hours of isolation and 10,000 cells were injected the next day subcutaneously into the flanks of mice. N-Myc alone generated a single tumor in 1 out of 16 mice with the resulting tumor being uniformly positive for the neuronal markers synaptophsyon, MAP2 and tyrosine hydroxylase (TH). This tumor also contained neuropil and ganglion cells and was characterized as neuroblastoma, differentiating subtype, stroma poor. 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% penetrance with 19 out of 19 mice generating tumors and with equivalent frequency in nude or syngeneic C57BL/6 mice showing this approach can be used in immune competent mice. The resulting tumors from this genetic combination were characterized as primitive neuroectodermal tumors with divergent differentiation. Regions of these tumors that were diffusely positive for neuronal markers appeared neuroblastoma-like or PNET-like. Osteosarcoma was also a prominent feature in every tumor with loss of p53. Additionally we observed yet to be defined tumor types that were negative by IHC for neuronal markers, Schwannian markers and melanoma markers. Generally, in mice with the fastest tumor onset micrometastases to the lung and liver were observed, while slower onset correlated with macroscopic metastases to the same organs. Injection of cell lines derived from primary tumors lead to aggressive metastatic spread to lung and liver. Though subcutaneous tumors rarely metastasize this was a salient feature of our neural crest derived cancers, which recapitulates the highly metastatic nature of human NCC derived cancers like neuroblastoma and melanoma. N-Myc in a wild type background caused a single phenotypically accurate neuroblastoma while loss of p53 caused an increase in cancer divergence underscoring the capacity of NCCs to generate a diverse array of cancer types including osteosarcoma. From this study we believe primary NCCs can be used as a platform for identifying initiating cancer events in neuroblastoma and possibly other pediatric malignancies. Citation Format: Rachelle R. Olsen, Otero Joel, Kirby Wallace, Jerold Rehg, Kevin W. Freeman. Modeling pediatric malignancies by transforming primary neural crest cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A04.