Only a small fraction of tumor cells is capable of passing through connective tissue barriers and forming metastatic foci. A quantitative in vitro tumor invasion assay using human amnion has made it possible to assess how external factors as well as normal cellular functions affect the invasive behavior of tumor cells. M5076 cells, derived from murine reticulum cell sarcoma, pass spontaneously through the amnion in significant numbers within 24 hours. When protein synthesis is blocked with cycloheximide, the number of invasive M5076 cells is reduced 82%. On the contrary, inhibition of DNA synthesis and cell proliferation has no effect on their invasiveness. The present project is focused on the genetic mechanism by which tumor cells penetrate connective tissue barriers. In an attempt to identify the genes responsible for the invasive behavior, NIH 3T3 cells and amnion epithelial cells were transfected with purified whole DNA extracted from various tumor cell lines. Tumors were formed in nude mice inoculated with 3T3 cells transfected with DNA from murine reticulum cell sarcoma (M5076) and DNA from human breast carcinoma (MCF-7). The in vitro amnion system is being used to select for transformed invasive cells. A second approach to examine whether the invasive behavior can be genetically transmitted is to use fused cell lines. Tumor cells of high (F10 melanoma) and low (2237 fibrosarcoma) metastatic potential were fused with benign neoplastic cells and normal cells. The present work includes comparing invasiveness and proteolytic activity of the fused lines with the parent lines. Besides using the amnion BM as an in vitro invasion model, it can be used as a growth substrate. Human and bovine endothelial cells grow well on the BM. Normal rat hepatocytes attach much better to the amnion BM than to any of the five types of collagen tested. We propose that this native BM substrate be used to study differentiation of various epithelial cells which normally reside on basement membranes.