Activation of Cancer Cell-Derived MMP-9 Correlates with Cancer Invasion- an in vitro study with 3-dimensional co-culture system-

Abstract

The metastasis of squamous cell carcinoma (SCC) as it invades surrounding tissue is a complex process that requires cancer cells to interact with stromal fibroblasts. During that interaction, cancer cells cause stromal fibroblasts to secrete and activate matrix metalloproteinases that degrade the extracellular matrix. Our experimental goal is to clarify the mechanisms by which MMP-9 is expressed and activated as SCC cells invade adjacent tissue. A human oral SCC cell line (HSC-3) and an embryonal lung fibroblast cell line (MRC-5) were co-cultivated in type I collagen gel. The expression and activation of MMPs in the culture supernatants were detected by gelatin zymography and Western blot analysis. We histologically examined the pattern of cancer-cell invasion in specimens harvested from our in vitro invasion model. Gelatin zymography and Western blot analyses showed that MMP-9 and MMP-2 were secreted by HSC- 3 and MRC-5, respectively. When HSC-3 cells were co-cultured with MRC-5 in the in vitro invasion model, pro-MMP-9 (92 kDa) was converted to the activated form (67 kDa), and we observed HSC-3 cells growing invasively into the collagen gel. In contrast, when HSC-3 cells were cocultivated with MRC-5 fixed with 2.5% glutaraldehyde solution, pro-MMP-9 was not activated, and no invasion of HSC-3 cells was observed. Cancer cell invasion requires the presence of fibroblast-derived factors that plays an important role in fully activating MMP-9, and we showed that MRC-5 fibroblasts secrete MMP-3, which is a potent MMP-9 activator, in this co-culture system.