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Selection and comparative analysis of novel prostate carcinoma dissemination variants : roles in metastasis of tumor-derived pro-uPA and neutrophil-derived MMP-9


In order to identify molecules and processes involved in carcinoma cell dissemination, novel prostate carcinoma variants have been isolated from the PC-3 parental cell line, which differ substantially in their ability to spontaneously metastasize. Comparative analyses of the resultant low and high disseminating variants, i.e., PC-lo /diss and PC-hi/diss, have demonstrated that the variants differ specifically in their capacity to complete early metastatic events. Concomitantly, the PC-lo/diss and PC-hi /diss cells exhibit respectively low and high angiogenic potential and differential abilities to escape from the primary tumor mass and invade extracellular matrix components in vitro and in vivo. Since extracellular proteolysis is required for tumor angiogenesis, invasion, and metastasis, levels of proteolytic enzymes were compared between the PC-lo/diss and PC-hi/diss variants, with an emphasis on matrix metallo- and extracellular serine proteases. These analyses highlighted elevated levels of the serine protease uPA and plasmin-generating capacity of PC-hi/diss cells. To mechanistically analyze the role of uPA in early dissemination of tumor cells, we prevented pro-uPA activation with function-blocking mAb- 112 or inhibited the activity of uPA-generated plasmin with aprotinin in a series of in vitro and in vivo models. Inhibition of uPA activation substantially reduced PC-hi/ diss spontaneous metastasis in chick embryo and orthotopic murine models. Furthermore, both angiogenesis and invasion were sensitive to mAb-112 and aprotinin treatments, indicating that uPA and uPA-generated plasmin exert their effects early in the metastatic cascade by facilitating primary tumor escape and local invasion of PC-hi/diss cells and by promoting tumor angiogenesis. While no major differences in tumor-cell derived matrix metalloproteinases were observed between the PC-3 variants, PC-hi/diss cells exhibited increased capacity to recruit host MMP-9 delivering neutrophils to primary tumors in avian CAM and murine orthotopic xenograft models. Inhibition of neutrophil influx into primary CAM tumors was achieved by neutralizing the potent neutrophil chemoattractant, IL-8. This treatment coordinately decreased levels of angiogenesis and metastasis, both of which could be rescued by exogenous TIMP-free neutrophil MMP-9, but not by MMP-9 complexed to TIMP-1. These findings highlight a significant angiogenic role for TIMP- free MMP-9 and indicate that aggressive tumor cells can efficiently recruit host MMP-9 delivering leukocytes to facilitate tumor progression. Overall, the comparative analyses of the novel PC-3 dissemination variants described herein demonstrate the importance of both tumor and inflammatory derived proteases in tumor dissemination. Furthermore, tumor-derived uPA and neutrophil-derived MMP- 9 regulate levels of angiogenesis concomitant with levels of metastasis, strongly implicating angiogenic blood vessels in directly facilitating tumor cell intravasation and dissemination

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