Solubilization of immobilized protein substrates by metastatic tumor cells

A new, sensitive automated assay based on the enzyme-linked immunosorbent assay was developed for measuring proteolytic enzyme activity produced by metastatic tumor cells. In this assay, a suitable protein substrate is adsorbed onto the surface of microplates and incubated with dilutions of standard proteinases, viable tumor cells, or tumor-cell-conditioned medium. The loss of immobilized protein due to proteolysis is then detected by means of antibodies directed against the target protein, and is measured by a microplate reader. Both casein and fibronectin were useful as substrates for the assay of various well-defined proteinases. The assay was successfully used to detect degradative activity elaborated by the mouse B16-BL6 melanoma and the human HT1080 fibrosarcoma cell lines. Both cell lines extensively removed immobilized protein substrates when the cells were seeded directly on the protein films. In addition, substrate removal could also be detected in the serum-free culture medium conditioned by the tumor cells. The results indicate that soluble proteinases secreted by tumor cells may be important during tissue invasion.