A Fluorescent Orthotopic Mouse Model for Reliable Measurement and Genetic Modulation of Human Neuroblastoma Metastasis

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Abstract
Neuroblastoma is the most common extra-cranial solid tumor of infancy and childhood, and majority of patients die from the metastatic disease. Orthotopic xenograft mouse models are valuable tools for improving our understanding and control of neuroblastoma metastasis, because they readily represent genetic diversity and allow spontaneous metastasis. Intra-adrenal injection is commonly used for establishing the orthotopic animal models since human neuroblastoma frequently originates in the adrenal gland. However, it is unclear whether the metastatic potential of neuroblastoma can be reliably determined in adrenally-injected mice because their gland size is so small. In this study, we developed and characterized a fluorescent orthotopic xenograft animal model of NB69-derived human neuroblastoma. By comparing animals receiving adrenal injection and adrenal overlay, with the latter mimicking injection spillover, we found that the metastatic potential of neuroblastoma can be reliably determined in animal lungs. Furthermore, the lung metastasis can be genetically modulated in these animals. The results also show that the expression of Renillagreen fluorescent protein (GFP) was exceptionally stable in NB69 cells, allowing rapid and sensitive detection of lung metastases at the macroscopic level. Additional features of our model include 100 tumor take, a 1-week tumor latency, resemblance to tumor behaviors in neuroblastoma patients, and the ability to monitor the expression of a gene of interest with GFP. This animal model of human neuroblastoma will be useful for studying genes involved in the metastatic process and for evaluating anti-metastasis agents in pre-clinical trials.