Imaging proteolysis by living human glioma cells

Degradation of basement membrane is an essential step for tumor invasion. I n order to study degradation in real time as well as localize the site of p roteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV c ollagen (DQ-collagen IV). Accumulation of fluorescent products is imaged wi th a confocal microscope and localized by optically sectioning both the cel ls 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 e arly as 24 hours after plating as monolayers, U87 cells were present throug hout the 3-dimensional matrix. Cells at all levels had accumulated fluoresc ent 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 ce lls and those within the spheroid contained fluorescent degradation product s of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells lik e breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.