Abstract The MET tyrosine kinase receptor is important during human development for normal cell growth and migration. Activating alterations of MET have also been identified in several carcinomas. Previous immunohistochemistry studies in our laboratory found high levels of MET in 85% of osteosarcoma tumor samples. In addition, pharmacologic modulation of MET resulted in a migratory decrease in some osteosarcoma cell lines. This led us to hypothesize that MET is necessary for osteosarcoma metastasis, and genetic down-regulation of MET expression would reduce the metastatic phenotype of osteosarcoma cells. We used lentiviral shRNA constructs to knockdown MET expression in several osteosarcoma cell lines. These included MNNG-HOS, a metastatic chemically transformed line that expresses constitutively activated MET, MG63.2, a highly metastatic variant of poorly metastatic MG63, AI and LR, which are two newly derived cell lines that have extremely high MET levels. We quantified levels of MET expression using a sensitive and quantitative electrochemiluminescence (ECL) assay and assessed in vitro parameters of metastasis including motility, migration, and invasion. MET expression was assessed by ECL in protein lysates from frozen osteosarcoma tumor samples and cell lines, and 15-20% of samples showed extremely high levels of total MET. AI, LR and MG63.2 had the highest levels of MET expression. MNNG-HOS has activation of MET due to translocation of the 3′end of MET with the 5′end of TPR. We utilized these lines for shRNA knock down of MET. ECL quantification of total MET levels revealed greater than 70% decrease in MET expression with shRNA knockdown in all four cell lines, compared with cells infected with scramble shRNA controls. As predicted for MNNG-HOS, shRNA constructs targeting the 3’ tyrosine kinase domain of MET resulted in marked decreased motility, compared to intermediate decreases using shRNA constructs targeting the 5’ region. Knock down of MET in MG63.2 resulted in inhibition of both motility and migration. AI and LR assays are in progress. Taken together, these data are the first evidence that genetic down-regulation of MET results in a decreased metastatic phenotype in osteosarcoma. This is a promising finding, as several small molecule tyrosine kinase inhibitors targeting MET are in development. We are currently testing our hypothesis in a mouse model to determine the ability of the above genetically knocked-down MET cell lines to decrease metastasis in vivo. Concurrently, in preparation for translational studies, we are also assessing MET specific tyrosine kinase inhibitors utilizing the parental cell lines described above. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3401.