Neurobtastoma is an enigmatic tumor, in which some patients who have widespread disease do well, even with no therapy, while others relentlessly progress, in spite of myeloablative therapy. A key clinical feature associated with bad outcome in neurobtastoma is the presence of bone metastases. In spite of the association of bone metastases with poor outcome in neurobtastoma, and the important contribution that bony disease makes to end-of-life suffering, there have been a paucity of biological studies on neurobtastoma bone metastases, and no clinical tdals. We propose to utilize our large (> 200) panel of human neuroblastoma cell lines to identify cell lines, which readily form metastases in bones of immunocompromised (SCID) mice. We will employ high-resolution small animal radiographic imaging to monitor the animals for bone metastases, and to identify sites for directed histopathology and cell culture at the time of necropsy. We then propose to use Affymetrix otigonucleotide array gene expression profiling in a highly selected panel of cell lines (6 shown to readily form bone metastases and 6 that do not when tested by i.v. injection into SCID mice) to identify genes in which altered expression (especially over-expression) correlates with the ability to form bone metastases in SCID mice. We will select for cells with an increased predilection to form bone metastases by repeated cycles of intravenous injection into SCID mice and harvest and re-culture of cells found growing in bone, and we will compare the selected cell lines to the original parental lines, and to cell lines that do not """"""""home"""""""" to bone, using Affymetrix expression profiling. We will confirm a relationship between gene over-expression (or under-expression) and bone metastases in our large panel of cell lines by quantitative RT-PCR for genes identified by expression profiling. RT-PCR will be conducted for all cell lines in the panel known to form or to not form bone metastases, and also will be conducted using xenograft tumors from mouse bone and compared to non-bone metastasizing xenografts grown subcutaneously or in other non-bony sites. This project will provide cell lines and animal models that will be useful for understanding the biology of bone metastases in neuroblastoma, and also for pre-clinical therapeutic studies. We will also identify candidate genes that are key for bone metastases in neuroblastoma. In future studies, beyond the scope of this proposal, we and/or other investigators can determine the ability of the identified candidate genes, when over expressed via transduction, to confer an ability to form bone metastases in SCID mice to cell lines known to rarely form bone metastases. Those candidate genes are likely to be applicable to tumors other than neuroblastoma. We anticipate that this project will increase our understanding of the biology of bone metastases and will develop mouse models for pre-clinical testing of new therapeutic approaches to high risk neuroblastoma.
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