Imaging proteolysis by living human breast cancer cells

Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through th e use of quenched-fluorescent protein substrates, Degradation thus was imag ed, by confocal optical sectioning, as an accumulation of fluorescent produ cts. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluore scence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellu lar fluorescence was observed in the BT549 cells in the presence of cytocha lasin B, suggesting that degradation occurred intracellularly and was depen dent on endocytic uptake of substrate. In the presence of a cathepsin B-sel ective cysteine protease inhibitor, intracellular fluorescence was decrease d similar to 90% and pericellular fluorescence decreased 67% to 96%, depend ing on the protein substrate. Matrix metallo protease inhibitors reduced pe ricellular fluorescence similar to 50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix protei ns by living human breast cancer cells, and 2) the cysteine protease cathep sin B participates in both pericellular and intracellular digestion of matr ix proteins by living human breast cancer cells.