UC San Diego

Intravasation, the process by which tumor cells enter the vasculature, is a rate-limiting step within the metastatic cascade. Matrix metalloproteinases (MMPs), an enzyme family of endopeptidases, are known to play roles in metastasis and angiogenesis, but the specific functions of MMPs during tumor cell intravasation remain unclear. To investigate molecular mechanisms of tumor cell intravasation, we have utilized high and low disseminating cell variants isolated from human HT-1080 fibrosarcoma, HT -hi/diss and HT-lo/diss respectively, which differ by 50 - 100-fold in their ability to intravasate and metastasize in the chick embryo. HT-1080 tumor cell variants form primary tumors on the chorioallantoic membrane (CAM) of chick embryos, and the cells escaping the primary site disseminate through the circulation. The escaped tumor cells arrest in the CAM, which serve as a repository for intravasated cells, and also metastasize to internal organs of the embryo. The disseminated human tumor cells are quantified by Alu qPCR. In this study, an RNAi approach to downregulate individual tumor MMPs was used to determine their functional roles in HT-hi/diss intravasation. MMP gene and protein expression were compared in HT-hi/diss and HT-lo/diss in culture and in primary tumors by qRT-PCR and Western blot analyses. Protein expression analysis of primary CAM tumors demonstrated that MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, MMP-2 was more abundant in the HT- lo/diss variant, and MMP-14 expression was similar in both variants. RNAi-mediated downregulation of three secretory MMPs, i.e. MMP-1, MMP-2 and MMP-9, substantially increased HT-hi/diss dissemination, suggesting protective roles for these enzymes in intravasation. Further investigation indicated that the enzymatic activity of tumor MMP-9 was needed to confer its protective role in intravasation. Moreover, tumor-derived MMP-9 exhibited pro- or anti- metastatic roles depending on cancer type since downregulation of MMP-9 resulted in opposite effects on HT -1080 fibrosarcoma and HEp3 carcinoma. Collectively, these findings demonstrated that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. catalytically interfering with the early stages of tumor dissemination. Therefore, targeting certain tumor MMPs in specific cancer types may result in enhanced malignancy