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The metastatic process is a dynamic interaction between malignant cells and the invading microenviroment. Neoplastic cells continue to grow by altering the local stroma and recruiting blood vessels through production and secretion of various growth factors and cytokines. The surrounding extracellular matrix reciprocates by providing a conducive microenvironment for these invading cells. Included among the secreted factors are matrix metalloproteinases and their natural inhibitors (TIMPs). Previously, we have shown that overexpression of TIMP-1 in H2009, a lung adenocarcinoma cell line yields HB-1, a highly aggressive cell line, both in vitro and in vivo. Implantation of these cells in the mouse CNS had resulted in larger and more frequent tumors by HB1 compared to H2009. Gene array analysis of H2009 and HB-1 cells had shown, among other factors, a 3-fold reduction in Thrombospondin-1 (TSP-1) levels. This was reflected in more aggressive proliferation and tube formation by endothelial cells in the presence of conditioned media from HB-1 compared to media from H2009.

In the present study, we have utilized siRNA methodology in an attempt to further delineate the role of TSP-1. Vector expressing siRNA to TSP-1 was tranfected into H2009 and stable clones (#1 and #8) were selected. Western blot analysis showed greatly reduced level of TSP-1 protein in these two siTSP clones, compared to H2009 and even HB-1. In vitro assessment of tumorigenicity as in a soft agar assay evaluating anchorage independent proliferation revealed that colony formation by siTSP clones was comparable to the parental cell line H2009, whereas HB-1 behaved more aggressively forming greater numbers of colonies. Preliminary studies assessing angiogenic responses in the Matrigel endothelial cell assay identified increased proliferation and complex connections in HB-1 and siTSP clones. These data show that, as anticipated, silencing TSP-1, a known inhibitor of angiogenesis, does not affect cell proliferation or invasion, however angiogenic responses are increased.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA