An in vitro cell invasion assay: Determination of cell surface proteolytic activity that degrades extracellular matrix

Abstract

A correlative morphological and biochemical method is described that facilitates assaying the invasive phenotype of tumor cells in vitro. The method involves a labeled matrix substrata to measure the activity of cell surface proteases, and uses the covalent linkage of fluorescence-labeled fibronectin (or other purified extracellular matrix components) to the surface of crosslinked gelatin substrata. We have investigated the invasiveness of tissue culture cells based on two criteria: the ability of the cells to form invadopodia and create surface indentations into crosslinked gelatin films that can be visualized by differential interference contrast microscopy and transmission electron microscopy, as well as their ability to locally degrade fibronectin or other matrix components coated on loosely crosslinked gelatin films by fluorescence microscopy. In using this method, we have demonstrated the existence of cell surface proteases that may be important in the invasion of cells into the extracellular matrix. Also, we have identified various cell types including embryonic fibroblasts transformed by Rous sarcoma virus, malignant human melanoma cell lines, breast carcinoma cell lines, neural microglia, and neural crest cells that are invasive in culture.