Live-cell proteolysis assay revealed disparate proteolytic patterns in 3D cultures of SUM149 and SUM190 cells. SUM149 and SUM190 cells were seeded onto glass-coverslips coated with reconstituted basement membrane containing DQ-collagen IV. Following overnight incubation at 37°C, cells and DQ-collagen IV cleavage products (green) were imaged using confocal microscopy (see Materials and Methods). 3D structures of live SUM149 and SUM190 cells degraded DQ-collagen IV pericellularly (arrowhead) and predominantly intracellularly (arrow), respectively. Confocal Z stack images were reconstructed in 3D [see Additional files 1 and 2] utilizing Volocity™ software (PerkinElmer, Waltham, MA, USA). Representative merged images of DIC and green channels of the equatorial plane (middle) and planes equidistant, 20 μm, from the equatorial plane (top and bottom) are also shown. Bar, 20 μm.