We have previously shown that interactions of stromal cells with breast and colon tumor cells enhance degradation of extracellular matrix proteins. To further study these interactions and the associated proteolysis, we have developed a 4D (3D + time) confocal/multiphoton microscopy assay utilizing quenched fluorescent DQ collagen IV™ substrate in Matrigel under live cell conditions. The cells used for these studies were BT20 and BT549 human breast carcinoma cells and HCT116 human colon carcinoma cells alone or in co-culture with WS-12Ti human breast fibroblasts and CCD-112CON human colon fibroblasts, respectively, in the presence and absence of protease inhibitors. Fibroblasts were prestained with Cell Tracker Orange™ or Cell Tracker Far Red™ to distinguish them from tumor cells. BT549 and HCT116 cells were grown on Matrigel/DQ-collagen IV™ in the absence of stromal cells to assess the efficacy of proteolysis by tumor cells alone. Starting at one hour after seeding, live cells were imaged for 24 hours by confocal microscopy. In the first few hours, i.e., when BT549 and HCT116 cells were very motile, proteolysis of DQ-collagen IV™ was mostly intracellular, becoming pericellular as the tumor cells formed spheroids. In contrast, the proteolysis by BT20 cells was pericellular at all timepoints. Both formation of spheroids and invasiveness into Matrigel was enhanced when tumor cells were cocultured with fibroblasts. We quantified proteolysis of DQ-collagen IV™ by tumor cell/stromal cell cocultures and showed that proteolysis was reduced with broad-spectrum inhibitors of MMPs, cysteine and serine proteases. We also imaged the morphology of tumor cell-fibroblast cocultures at longer times, i.e., after 2 weeks in culture. Fibroblasts were found to surround the disorganized acinar structures formed by BT20 cells and aggregates formed by BT549 cells. In both cases, invasion of the tumor cells through Matrigel was markedly increased in the 2 week cocultures. Our findings support the contention that host stromal cells contribute significantly to overall tumor proteolysis and that more than one catalytic-type of protease is involved in this process.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]