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Fig. 1. Tumor cells induce MMP-9 expression in fibroblasts. (A) Morphology of a coculture of DFs and CA1a cells. Spindle-shaped fibroblasts (S) surround tumor-cell islets (T). Bar, 200 µm (B) (a) An additional MMP which runs slightly higher on SDS-PAGE (as shown by gelatin zymography) than human MMP-9 was induced in cocultures. (b) Activity of the induced gelatinolytic enzyme was blocked by the addition of 10 mM EDTA to the developing buffer during gelatin zymography. Tissue-culture supernatants used in (a) were taken from cocultures, for (b) they were taken from cultures grown in a Millipore system that separates CA1a cells from DFs by a membrane, which allows exchange of signals but prevents cell-cell contact. (C) In DFs, the gelatinolytic activity was induced with medium conditioned by CA1A cells. Media, conditioned for 4 days by DFs or CA1A cells, were used to stimulate homotypic cultures of DFs or CA1A cells (lanes labeled CA1a and DF). As a negative control, cells were stimulated with media that had been incubated for 4 days in an empty dish to distinguish between the influence of CA1A cells and DFs on the media and the age of the medium (4-day incubation at 37°C) (lanes labeled medium). (D) Gelatin zymography of the tissue-culture supernatant and the pellet obtained by immunoprecipitation. The induced enzyme was removed from the supernatant by immunoprecipitation with an anti-MMP9 antibody but not an anti-MMP2 antibody. (E) The gelatinolytic enzyme was not induced in the medium of CA1A and MMP9KO cells cocultured for 4 days, but was induced in cocultures of CA1A and MMP2KO cells and also in cocultures of CA1A cells and the corresponding wild-type fibroblasts MMP9WT or MMP2WT. Homotypic cultures and also medium kept in empty dishes served as controls.
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