INVESTIGATION OF GENETIC DETERMINANTS OF METASTATSIS IN A MURINE MODEL OF OSTEOSARCOMA WITH SPONTANEOUS METASTASIS USING cDNA MICRO-ARRAY

Khanna C, Khan J, Meltzer P, Helman L., (Pediatric Oncology Branch, National Cancer Institute, and Cancer Genetics Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892).

Osteosarcoma (OSA) is the most common primary tumor of bone. Despite successful control of the primary tumor and adjuvant chemotherapy, relapse of OSA in the lungs occurs in over 30% of patients within 5 years. In order to understand the complex process of metastasis from appendicular tumors to the lungs we have developed and applied two murine models of osteosarcoma differing in pulmonary metastatic potential to a murine cDNA micro-array.

The murine model is characterized by orthotopic primary tumor growth in balb/c mice, local tumor control with amputation, and spontaneous pulmonary metastases. Using cell lines described by Schmidt et al, 1983 (K-12) and a novel cell line (K-7M2) derived from the model, low and high metastatic models, respectively, were defined. The K-12 model is characterized by pulmonary metastases in 33% of mice compared to over 90% in the K-7M2 model.

The cDNA micro-array slides and analysis system were developed in the Human Genome Research Institute. Probe and slide preparation, probe hybridization, and analysis have been previously described (Khan et al, 1998). RNA was purified from the primary tumors and from the respective tissue culture cell lines. Array comparisons consisted of primary tumor extracted RNA (K-12 versus K-7M2) and in separate experiments tumor cell line extracted RNA (K-12 versus K-7M2). Analysis allowed the relative expression of 2200 murine cDNAs in K-12 and K-7M2 to be evaluated. A 2 fold difference in expression was used to define genes as over- expressed.

Comparison of K-12 versus K-7M2 yielded 73 genes over-expressed in the primary tumor RNA, 39 genes in cell culture RNA, and 12 genes in both tumor and cell culture RNA. Comparison of K-7M2 versus K-12 yielded 33 genes over-expressed in the primary tumor RNA, 26 genes in cell culture RNA, and 10 genes in both tumor and cell culture RNA. The following genes found over-expressed have been confirmed for identity by sequence analysis at this time.

K-12 over-expressed genes: osteocalcin precursor, PPAR-gamma, thrombomodulin, S- Protein, complement factor H, and slug.

K-7M2 over-expressed genes: integrin beta4 subunit, GDP dissociation inhibitor-l, macrophage cysteine rich transmembrane glycoprotein, clusterin, and ezrin. Genes identified include those suggesting greater bone and mesenchymal differentiation in the less aggressive K-12 model (osteocalcin, PPAR-gamma), over-expression of genes with roles in metastasis in K-7M2 (integrin beta4 subunit), and genes with functions that may be associated with metastasis or suppression of metastasis; although, not previously described in OSA (slug, clusterin, ezrin). Using this model system the biological relevance of these genes to the process of metastasis will be investigated. The relevance of these genes in other models of OSA and in human OSA will also be examined.