1.15 Establishment of an Orthotopic Xenograft Model of Metastatic Neuroblastoma Using Ultrasound Guidance

R. Van Noord1, D. Bashllari1, M. Hoenerhoff2, E. A. Newman1  1University Of Michigan,Pediatric Surgery,Ann Arbor, MI, USA 2University Of Michigan,In Vivo Animal Core, Pathology Core,Ann Arbor, MI, USA

Introduction:  Neuroblastoma (NB), the most common cancer in infancy arises from neural crest precursors in the adrenal medulla and sympathetic neural ganglia. The majority of tumors arise in the adrenal gland. Nearly 50% of patients have high-risk Stage IV disease that relapses after standard multimodality therapy.  In order to better understand NB tumor biology and to examine novel therapeutic approaches, cancer researchers often utilize animal xenograft models.  Surgical models are often tedious and labor intense with pain and long recovery periods.  Further limitations of previous models include slow rate of tumor formation, inappropriate microenvironment, and lack of metastases. We hypothesized that a minimally invasive technique utilizing high-resolution ultrasound guided needle injections of tumor is a safe and efficient method to establish orthotopic xenograft models of NB for preclinical studies. 

Methods: Real-time ultrasound procedures were performed utilizing the VisualSonics’ Vevo 770 in vivo imaging system. Six week-old immune deficient mice (NOD-SCID) were anesthetized in an induction chamber. A chilled Hamilton syringe fitted with a 27 G needle loaded with luciferase-tagged human NB cells (SH-SY5Y or IMR-32) suspended in matrigel was guided into the adrenal gland capsule utilizing ultrasound guidance.  Cell suspensions were hand injected into the adrenal gland, above the kidney and below the liver or spleen. Tumor growth was measured weekly via bioluminescence.  Tumors were analyzed by veterinary science pathologists in the Pathology Cores for Animal Research, and characterized using paraffin-embedded formalin-fixed slides stained for H/E, neuron specific enolase, and tyrosine hydroxylase. A total of 18 animals were studied (SH-SY5Y and IMR-32).  All procedures were followed and approved by the University Committee on Use and Care of Animals.

Results: There was minimal animal recovery post-procedure, and no morbidity or mortality. In all animals, NB tumors successfully engrafted at the suprarenal space confirmed by weekly imaging via bioluminescence and histological characterization.  There was evidence of local tumor engraftment by seven days post-injection.  Tumors progressed to bulky local disease with distant metastases by four weeks.  Metastatic sites included lymph nodes and bone, sites typical of NB spread.

Conclusions: We demonstrate that a novel metastatic murine model of NB is established by a percutaneous, ultrasound guided technique. Tumors are detected, monitored and quantified prior to visualization using bioluminescence.  This minimally invasive technique is target specific, safe, and efficient, making it an optimal metastatic preclinical model to test new therapeutic agents for high-risk neuroblastoma.