The long term goal of this work was to develop an in vivo model of metastasis in which it would be possible to a) quantitatively analyze the individual steps of this process, b) to identify tumor cell properties which are crucial for their spread, and c) to determine in what way the alteration of these properties affects metastasis. the latter would provide an insight to the mechanism of tumor cell spread and, possibly, a target for therapy. the work is centered on proteolytic enzymes, such as urokinase type plasminogen activator (uPA), plasmin and collagenases, which are able to degrade biological barriers and thus may facilitate the escape of cells from a primary tumor and their dissemination to distant sites. Using chick embryos and human tumor cells we developed an assay in which """"""""spontaneous"""""""" metastasis, local invasion and extravasation (exit from the blood vessels) could be quantitated. We established that uPA had a crucial role in metastasis and that at least one step, the local invasion, was dependent on active uPA. Moreover, we showed that only cells which, in addition to uPA, also expressed surface receptor for uPA (uPAR) were locally invasive. By indirect means we also showed that interstitial collagenase (IC) may be involved in invasion. In the current proposal we will adapt the model to the quantitative study of invasion of blood vessels walls, which, to the best of our knowledge, will be the first such in vivo assay. Once developed, we will explore the possibility of adapting it in the future for testing the blood vessel invading capacity of fresh human tumors. The main emphasis of the current proposal is on direct examination of the uPAR and IC roles in the processes leading to metastasis; uPAR will be studied both for its capacity to concentrate proteolytic activity on the surface of tumor cells, and also as a possible mediator of enhanced tumor cell motility. These studies will be conducted in two hosts, chick embryos and nude mice. uPAR and IC will be inhibited in two malignant human carcinoma cells (HEp3 and Colo 16) by antisense RNA. These cells will be stably transfected by constructs containing fragments of uPAR and IC cDNAs in antisense orientation; appropriate controls will be included. Cells inhibited in a single, defined property, will be tested for their ability to complete individual steps of metastasis. This approach should enable the identification of tumor cells properties which cannot be compensated by alternative pathways and thus are rate limiting for the entire process. The mechanism(s) by which the metastatic process is interrupted will also be explored.
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