Preclinical Models for Neuroblastoma: Establishing a Baseline for Treatment

Abstract

BackgroundPreclinical models of pediatric cancers are essential for testing new chemotherapeutic combinations for clinical trials. The most widely used genetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse. This neuroblastoma-prone mouse recapitulates many of the features of human neuroblastoma. Limitations of this model include the low frequency of bone marrow metastasis, the lack of information on whether the gene expression patterns in this system parallels human neuroblastomas, the relatively slow rate of tumor formation and variability in tumor penetrance on different genetic backgrounds. As an alternative, preclinical studies are frequently performed using human cell lines xenografted into immunocompromised mice, either as flank implant or orthtotopically. Drawbacks of this system include the use of cell lines that have been in culture for years, the inappropriate microenvironment of the flank or difficult, time consuming surgery for orthotopic transplants and the absence of an intact immune system.Principal FindingsHere we characterize and optimize both systems to increase their utility for preclinical studies. We show that TH-MYCN mice develop tumors in the paraspinal ganglia, but not in the adrenal, with cellular and gene expression patterns similar to human NB. In addition, we present a new ultrasound guided, minimally invasive orthotopic xenograft method. This injection technique is rapid, provides accurate targeting of the injected cells and leads to efficient engraftment. We also demonstrate that tumors can be detected, monitored and quantified prior to visualization using ultrasound, MRI and bioluminescence. Finally we develop and test a “standard of care” chemotherapy regimen. This protocol, which is based on current treatments for neuroblastoma, provides a baseline for comparison of new therapeutic agents.SignificanceThe studies suggest that use of both the TH-NMYC model of neuroblastoma and the orthotopic xenograft model provide the optimal combination for testing new chemotherapies for this devastating childhood cancer.

Endoplasmic Reticulum (ER) Stress and Hypoxia Response Pathways Interact to Potentiate Hypoxia-inducible Factor 1 (HIF-1) Transcriptional Activity on Targets Like Vascular Endothelial Growth Factor (VEGF)

“…Cell Culture-The human neuroblastoma cell lines NB1691 and SK-N-AS were cultured as described previously (12). Lowglucose conditions were achieved by culturing cells in RPMI medium containing 1 mM glucose.…”

“…SK-N-AS cells were suspended in Matrigel (BD Worldwide) at a concentration of 2 ϫ 10 4 cells/liter. Para-adrenal injections were performed in CB17-SCID mice as described previously (12).…”

The Protumorigenic Role of Caspase-8 in Neuroblastoma

“…The CAM assay has been extremely valuable for the analysis of angiogenesis and metastasis; though not commonly used for tumor growth studies and comparisons. Our orthotopic xenograft model implicating a role for casp8 in promoting tumorigenesis was previously tested and validated to effectively mimic the expression of key hallmarks of the TH-MYCN mouse model and NB patient tumor samples [165]. No such studies have been published for NB tumor growth in the CAM assay.…”

“…To represent this event in a mouse model, we developed a novel, minimally invasive, ultrasound-guided orthotopic xenograft mouse model [165]. This method results in low morbidity and mortality, and has a high tumor engraftment success rate.…”

“…This method results in low morbidity and mortality, and has a high tumor engraftment success rate. Histological examination of xenografts derived from this orthotopic model expressed many of the key hallmarks of neuroblastoma including PGP 9.5, synaptophysin, neuron specific enolase, and tyrosine hydroxylase [165].…”