Abstract A19: Transformation of primary neural crest cells to model pediatric cancers
Authors: Olsen Rachelle R. , Otero Joel H. , Garcia-Lopez Jesus , Wallace Kirby A. , Yin Zhirong , Freeman Kevin W. 
Published: May 2016

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 &gt;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 the fastest onset of tumor growth displayed micrometastases to the lung and liver, while mice with slower tumor growth had macrometastases to these organs. Injection of cell lines derived from the primary tumors also lead to aggressive metastasis to the lung and liver. Although metastasis from subcutaneous tumors is generally rare, it was a recurrent feature in our model which recapitulates the highly metastatic nature of human cancers derived from NCCs like neuroblastoma and melanoma.

Lastly, we determined whether NCCs were sensitive to the BET bromodomain inhibitor JQ1. JQ1 treatment significantly inhibited growth of both primary NCCs and cell lines derived from NCC tumors, while having minimal effect on NIH-3T3 fibroblasts. Furthermore, our results imply that JQ1 sensitivity in these cells is independent of MYCN expression levels.

Using primary mouse NCCs, we found that MYCN overexpression is capable of initiating cell transformation and formed a single tumor with characteristics of neuroblastoma. Additional loss of p53 resulted in a more diverse panel of tumor types, which emphasizes the ability of NCCs to contribute to multiple lineages. Our results suggest that studies utilizing primary NCCs may be beneficial to identify initiating oncogenic events in neuroblastoma and perhaps other pediatric cancers.

Citation Format: Rachelle R. Olsen, Joel H. Otero, Jesus Garcia-Lopez, Kirby A. Wallace, Zhirong Yin, Kevin W. Freeman. Transformation of primary neural crest cells to model pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A19.