Imaging proteolysis by living human glioma cells.
Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.
Sameni
M
M
Department of Pharmacology and the Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Dosescu
J
J
Sloane
B F
BF
D002401:Cathepsin B / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005456:Fluorescent Dyes; D005910:Glioma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D018613:Microscopy, Confocal; D009361:Neoplasm Invasiveness; D010447:Peptide Hydrolases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D024141:Collagen Type IV; D005456:Fluorescent Dyes; D010447:Peptide Hydrolases; D002401:Cathepsin B