Th-MYCN Mice with Caspase-8 Deficiency Develop Advanced Neuroblastoma with Bone Marrow Metastasis

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow (BM). Advances in therapy and understanding of the metastatic process have been limited due in part, to the lack of animal models harboring BM disease. The widely employed transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the BM. This model recapitulates two frequent alterations in metastatic neuroblasoma, over-expression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. While over-expression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced BM metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix (ECM) structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation and down-regulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine transforming growth factor beta (TGF-β) pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood borne metastasis and therefore activation of this pathway may explain the enhanced BM metastasis observed in this animal model.Fil: Teitz, Tal. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Inoue, Madoka. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Valentine, Marcus B.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Zhu, Kejin. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Rehg, Jerold E.. St. Jude Children’s Research Hospital. Department of Pathology; Estados UnidosFil: Zhao, Wei. St. Jude Children’s Research Hospital. Department of Biostatistics; Estados UnidosFil: Finkelstein, David. St. Jude Children’s Research Hospital. Department of Computational Biology; Estados UnidosFil: Wang, Yong-Dong. St. Jude Children’s Research Hospital. Hartwell Center for Bioinformatics and Biotechnology; Estados UnidosFil: Johnson, Melissa D.. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Calabrese, Christopher. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Hakem, Razqallah. University of Toronto. Ontario Cancer Institute. Department of Medical Biophysics; CanadáFil: Weiss, William A.. University of California. Departments of Neurology, Pediatrics and Neurological Surgery; Estados UnidosFil: Lahti, Jill M.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados Unido

Preclinical Models for Neuroblastoma: Establishing a Baseline for Treatment

Preclinical 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.Here 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.The 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

Th-MYCN Mice with Caspase-8 Deficiency Develop Advanced Neuroblastoma with Bone Marrow Metastasis

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblastoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-β pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model