We prepared a specific antagonist for hepatocyte growth factor (HGF) and designated it HGF/NK4. HGF/NK4 is composed of N-terminal 447 amino acids of the K K-chain of HGF, thus contains the N-terminal hairpin domain and subsequent four kringle domains. HGF/NK4 competitively inhibited the specific binding of HGF to the receptor. Importantly, HGF/NK4 neither stimulated DNA synthesis of primary cultured rat hepatocytes (mitogenesis) nor induced cell scattering (motogenesis) and branching tubulogenesis (morphogenesis) of MDCK renal epithelial cells, however, HGF/NK4 almost completely inhibited the mitogenic, motogenic, and morphogenic activities of HGF. HGF/NK4 also suppressed tyrosine phosphorylation of the cMet/HGF receptor induced by HGF. Apparently this is the first documentation of a specific antagonist which abrogates the mitogenic, motogenic, and morphogenic activities of HGF.z 1997 Federation of European Biochemical Societies.

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which aects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal ®broblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-d1 cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.

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Growth and motility of carcinoma cells are regulated through their interactions with host stromal cells, i. e., tumor‐stromal interactions. Hepatocyte growth factor (HGF), a ligand for c‐Met tyrosine kinase, is a stromal‐derived regulator of growth, motility, and morphogenesis. HGF stimulated proliferation and motility of GB‐d1 gallbladder carcinoma cells from a patient with gallbladder cancer. HGF induced in vitro invasion of GB‐d1 cells into a collagen gel matrix, and this potent, invasive effect was not seen with epidermal growth factor, transforming growth factor‐β1, basic fibroblast growth factor, or platelet‐derived growth factor. Although GB‐d1 did not produce HGF, the cells did produce a factor which enhances HGF production in human skin fibroblasts, and this factor proved to be interleuldn‐1β (IL‐1β). When GB‐d1 cells were co‐cultured with fibroblasts such that a collagen gel matrix was layered between the GB‐d1 cells and fibroblasts, GB‐d1 cells invaded the gel, but invasion of the cells in the co‐culture system was inhibited by antibodies against HGF and partially inhibited by antibodies against IL‐1β. Thus, GB‐d1 cell‐derived IL‐1β stimulates HGF production in stromal fibroblasts and HGF up‐regulated in the fibroblasts induces invasion of GB‐d1 cells. The looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and a HGF‐inducer such as IL‐1β may be one mechanism which would explain the acquisition of malignant phenotype through tumor‐stromal interactions.

Hepatocyte growth factor (HGF), a ligand for Met tyrosine kinase, is a mesenchyme- or stroma-derived multipotent factor that regulates the growth, motility, and morphogenesis of various types of cells. During lung development, Met/HGF receptor mRNA was localized in lung epithelial cells, whereas HGF mRNA was localized in lung mesenchymal cells in rat embryos. Antisense HGF oligonucleotides specifically inhibited epithelial branching morphogenesis in cultured lung rudiment isolated from day-13 rat embryos, whereas recombinant HGF stimulated branching morphogenesis. Thus, HGF seems to be at least one of the mesenchyme-derived factors that support branching morphogenesis during lung development. Together with the finding that HGF plays important roles in organogenesis and morphogenesis of organs such as the liver and kidney, HGF seems to be a mediator in epithelium-mesenchyme interactions during organogenesis. Extending the conceptual framework of epithelium-mesenchyme (or epithelium-stroma) interactions, we next examined the possible involvement of HGF in tumor-stroma interactions, because the growth and motility of carcinoma cells are regulated through their interactions with host stromal cells. HGF induced in vitro migration and invasion of GB-d1 gallbladder carcinoma cells into basement membrane components and collagen-gel matrix; however, several other growth factors did not induce marked migration and invasion of the carcinoma cells. GB-d1 cells do not produce HGF, but they produce in inducing factor for HGF production in fibroblasts; the inducing molecule was identified as interleukin 1 beta. Cocultivation of GB-d1 cells with stromal fibroblasts embedded in a collagen-gel matrix induced invasion of GB-d1 cells into the collagen gels, but invasion was inhibited by a specific antibody against HGF. This indicates that in vitro invasion of GB-d1 cells depends on stromal fibroblasts and that the fibroblast-derived invasion factor is HGF. Since HGF stimulated in vitro migration and invasion of various carcinoma cells and several carcinoma cells produced inducing factors for HGF production in stromal fibroblasts, the looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and HGF inducers may be a mechanism responsible for acquisition of the malignant phenotype through tumor-stroma interactions.

Peritoneal injury enhances peritoneal implantation of carcinoma cells. Repair of injured peritoneum at trocar sites may reduce the frequency of wound metastases in laparoscopic surgery for unexpected gallbladder carcinoma.

To study the mechanism of invasion and metastasis of gallbladder cancer cells, we established a cancer cell line, GB‐d1, from a metastatic lymphnode of poorly differentiated adenocarcinoma of the gallbladder. GB‐d1 cells proliferate well in a dish culture and form small cystic cell clusters in a collagen gel containing 10% fetal bovine serum. A conditioned medium of human embryonic lung fibroblasts (HEL) stimulated the proliferation of GB‐d1 cells and induced cell scattering in the dish culture. In the gel culture, the conditioned medium induced a transformation of the spherical clusters to arborizating colonies with tubular projections that mimicked an invasion of cancer cells into the surrounding tissue. Similar results were obtained when 10 ng/ml of human recombinant hepatocyte growth factor (h‐rHGF) was added to the culture medium. The proliferative and morphological changes induced by the conditioned medium were inhibited by antiserum against h‐rHGF. HEL and human gallbladder stromal fibroblast‐like cells produced substantial levels of HGF in the culture media, while GB‐d1 did not produce any detectable level of HGF. These results suggest that HGF promotes the invasive growth of gallbladder cancer cells in vitro, and it was also suggested that stromal fibroblasts may play an important role in the invasive progression of gallbladder cancer in a paracrine fashion.

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