W. E. Barry1, J. R. Jackson1, G. Asuelime1, Z. Wan2, R. C. Seeger2, E. S. Kim1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Pediatrics,Los Angeles, CA, USA
Introduction: Neuroblastoma is the most common solid tumor in children and despite current multimodal therapies, survival is poor. While massive primary tumors are a clinical challenge for these children, the main cause of death is recurrent, incurable, metastatic disease following surgical resection. It is critical to further characterize the biologic differences between the primary tumor and the metastatic cells which appear to respond differently to treatment and are the major cause of mortality in these patients. We hypothesize that cells derived from liver and bone marrow metastases in a murine model will be phenotypically and genotypically different when compared to their parental cell of origin.
Methods: Multiple metastatic cell lines were created de novo from the bone marrow and liver of NSG mice utilizing two human neuroblastoma cell lines (CHLA 255, SH-SY5Y). A metastatic neuroblastoma mouse model of minimal residual disease was used whereby the parental cell line was injected into the renal capsule, followed by primary tumor resection 7 days later. The metastases were allowed to grow until euthanasia criteria were met. Metastatic neuroblastoma cells from the liver and bone marrow were harvested and cultured. In vitro studies comparing the parental cell line to the metastatic cell lines were performed. Response to increasing doses of chemotherapy (etoposide) was analyzed using a DIMSCAN cytotoxicity assay. Gene expression was characterized using a low-density metastatic pathway PCR microarray. The PCR-array was performed using the parental and metastatic cell lines from the SH-SY5Y cell line.
Results: Metastatic cell lines derived from the liver and bone marrow had a significantly lower response to increasing doses of etoposide (p<0.05) compared to the parental cell line, which followed a dose response curve. PCR array revealed numerous genes which were upregulated in the liver and bone marrow cell lines compared to the parental (Figure). Numerous neuroblastoma associated genes appeared to be upregulated including APC, CDH1, CXCL12, FGFR4, MET and NR4A3. These genes play critical roles in cell adhesion, migration, proliferation and survival. Many of the other upregulated genes were also vital to cell migration and adherence including CDH6 and MMP10.
Conclusion: Metastatic neuroblastoma cells appear to have significantly different gene expression as well as response to standard chemotherapy when compared to the parental cell line. These preliminary data provides the background for further in vitro and in vivo studies to elucidate the effects of treatments particularly on genes specific to metastatic disease in our novel murine model of minimal residual disease.
