Abstract C35: The development of metastatic mouse models of neuroblastoma to bone and brain to identify the molecular mechanisms governing metastasis
Authors: Fathers Kelly E. , Mouaaz Samar , Rioseco Constanza , Mourskaia Anna , Blasberg Ronald , Irwin Meredith , Siegel Peter , Kaplan David 
Published: August 2014

Abstract
Metastasis is a key reason for the lack of success in treating children with neuroblastoma (NB). Over 50% of patients present with metastatic lesions in the bone, brain, liver and lymph nodes, with a high fatality rate for patients that relapse with those metastases. A better understanding of the molecular processes defining the complexity and multi-step nature of NB dissemination is required for the development of effective treatments. To date, there is no NB model that quantitatively and reproducibly metastasizes to bone and brain. Thus, we developed novel metastatic mouse models of neuroblastoma to bone and brain through in vivo selection to enhance metastatic capability. SK-N-AS, derived from a bone marrow metastasis, and IMR-32, a MYCN-amplified line derived from an abdominal mass, were tagged for in vivo monitoring using a triple reporter (TR) encoding TK, GFP and firefly luciferase, which allows for nuclear imaging, FACs analysis, and whole body bioluminescence. Reporter expression was confirmed by double sorting for GFP and testing for luciferase activity. Cells were introduced into the blood stream of NOD/SCID mice through intra-cardiac injection, and monitored bi-weekly using bioluminescence. SK-N-ASTR cells metastasized to the adrenal gland (87%, 13/15 animals) and bone (mandible, hindlimbs, 67%, 10/15 animals), whereas IMR-32TR cells metastasized to bone (100%, 10/10 animals) and brain (40%, 4/10 animals). Metastatic lesions were confirmed by ex-vivo bioluminescence, 3D bioluminescence, MRI or micro-CT. Cells from specific metastatic sites of individual mice were isolated, expanded in culture, GFP sorted and injected into a second cohort of mice. Several of the SK-N-ASTR re-injected cell lines derived from bone exhibited enhanced metastasis after one round of in vivo selection, with 100% metastasis to bone and the adrenal gland (a common location of primary NB tumours) and up to 60% metastasis to brain, as confirmed by MRI. Subsequently, these animals had a decreased overall survival rate of approximately 43 days relative to the parental cell line, of 69 days. The metastatic sub-populations do not proliferate at higher rates than the parental cell line, as assessed by BrdU labeling. Thus, the enhanced metastasis observed in vivo is not due to a faster growth rate, but rather selection of metastasis promoting functions. This is supported by the finding that the metastatic SK-N-ASTR sub-populations also exhibited enhanced migration and invasion toward serum, as assessed using boyden chambers, compared to the parental line. These sub-populations are being characterized to identify the molecular mechanisms governing metastasis. Metastasis to brain and bone is an often fatal event in children with newly diagnosed and recurrent NB. We have developed the first reproducible and efficient NB metastatic mouse model to identify novel genes and signalling proteins regulating metastasis, and to identify drug candidates that suppress metastatic growth.
Citation Format: Kelly E. Fathers, Samar Mouaaz, Constanza Rioseco, Anna Mourskaia, Ronald Blasberg, Meredith Irwin, Peter Siegel, David Kaplan. The development of metastatic mouse models of neuroblastoma to bone and brain to identify the molecular mechanisms governing metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C35.