Fig. 4. In fibroblasts, TGF-ß, TNF-, EGF and HGF are involved in the induction of MMP-9 by tumor cells. (A) TGF-ß1, TNF- and, to a lesser extent, EGF induce the expression of MMP-9 in serum-starved (0.5% serum) DFs as shown by gelatin zymography (experimental set up as described in Fig. 3). (B) Indirect immunofluorescence and immunohistochemistry of cocultures of DFs and CA1a shows expression of TGF-ß mainly in tumor cells. The highest immunoreactivity for TNF- is observed in CA1a cells, migrating on the fibroblast monolayer (arrow heads). Bars, 32 µm (TGF-ß) and 100 mm (TNF-). (C,D) Neutralizing TNF- or HGF antibody significantly decreases MMP-9 levels induced by CA1a lysate. CA1a lysate and serum-starved (0.5% FCS), 80% confluent DFs were separately preincubated for 30 minutes with the antibodies, before combining and incubating them for an additional 24 hours. Cell-free culture-supernatants were analyzed by gelatin zymography. (E) Neutralizing HGF antibody slightly reduces levels of MMP-9 induced by TGF-ß1 (experimental set-up as in C). (F) MMP-9 induction by TNF- is decreased by neutralizing HGF antibody (experimental set-up as in C). (H) The EGF-receptor inhibitor AG1478 interfers with TGF-ß- but not TNF–induced MMP-9 induction (experimental set up as in Fig. 3E).