The stroma of hepatocellular carcinomas (HCC) is infiltrated with myofibroblasts (MFs). Preliminary in vivo data have suggested that liver MF express hepatocyte growth factor (HGF), a cytokine that has been implicated in several tumor models. Our aim was to investigate the role of MF and HGF in HCC. Cultured liver MF expressed HGF messenger RNA (mRNA) and secreted HGF in their medium, as shown by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Addition of MF-conditioned medium to the HepG2 HCC cell line induced cell scattering. This was associated with a decrease in cell proliferation. MF also increased about 100-fold the ability of HepG2 to invade Matrigel. Increased invasiveness was also shown for HuH7 cells, but no scattering was observed and cell proliferation was stimulated. All the effects of MF on both tumor cell types were blocked by addition of an antibody to HGF and they all could be reproduced by adding recombinant HGF to the tumor cells. RT-PCR and Western blot analysis confirmed that both tumor cell lines expressed c-met, the receptor for HGF. The effects of MF-conditioned medium were not reproduced by acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1), or platelet-derived growth factor (PDGF-BB). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that HGF was expressed in human HCC. Our data show that human liver MF act on HCC cells to increase their invasiveness and suggest that MF-derived HGF could be involved in the pathogenesis of HCC.

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RT-PCR and Western blot analysis confirmed thatAlso, tumor-associated MF have been shown to be involved both tumor cell lines expressed c-met, the receptor for HGF.in the in vivo and in vitro invasiveness of colon cancer cell The effects of MF-conditioned medium were not reproduced lines. 6 by acidic fibroblast growth factor, basic fibroblast growth Hepatocyte growth factor (HGF) is a multifunctional cytofactor, epidermal growth factor (EGF), transforming growth kine that has been implicated in the pathogenesis of several factor-b1 (TGF-b1), or platelet-derived growth factor (PDGFtumors. It acts on the cells via its specific receptor, c-met. 7 BB). Reverse transcription-polymerase chain reaction (RTIt has recently been shown, using in situ hybridization, that PCR) analysis confirmed that HGF was expressed in human human liver myofibroblasts express HGF in patients with HCC. Our data show that human liver MF act on HCC cells cirrhosis. 8 Although there are still some contradictory results, to increase their invasiveness and suggest that MF-derived most studies indicate that HGF is expressed in human, 9-13 or murine HCC. 14,15

We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII. Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.Hepatocellular carcinoma (HCC) 1 is one of the most frequent primary tumors in the world (1). It is a major complication of liver cirrhosis, although more rarely it will develop on a noncirrhotic liver. HCC are characterized by a high rate of local, intra-hepatic invasion. HCC are infiltrated by myofibroblastlike cells, located around tumoral sinusoids and in fibrous septa and capsule, when present (2-4). We have previously shown that cultured human liver myofibroblasts strongly promoted in vitro invasion of human HCC cell lines through their secretion of hepatocyte growth factor (HGF) (5). In further studies, we showed that HGF induced invasion by increasing the expression of the urokinase-type plasminogen activator (uPA) by the cancer cells (6). Indeed, myofibroblast-or HGFinduced invasion was dose-dependently blocked by a selective uPA antagonist (6). One of the main functions of uPA is to convert the inactive zymogen plasminogen into plasmin, a broad-spectrum proteinase able to degrade several components of the extracellular matrix and thus a likely effector of cancer cell invasion.Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5 is a serine proteinase inhibitor containing 3 tandemly arranged Kunitz-type proteinase inhibitor domains, homologous to tissue factor pathway inhibitor (7). TFPI-2 exists as 3 isoforms of 27, 31, and 33 kDa that are synthetic products of a single gene and arise from differential glycosylation. TFPI-2 is a strong inhibitor of plasmin as well as of trypsin, plasma kallikrein, and factor XIa. It does not inhibit uPA (8). TFPI-2 synthesis has been described in dermal fibroblasts and endothelial cells (9 -11). In these cell types, the major part of TFPI-2 is sequestered within the extracellular matrix (ECM), presumably bound to heparan sulfate. In the course of a systematic sequencing of a human liver myofibroblast cDNA library described elsewhere (12), we found that these cells expressed transcripts for TFPI-2. Given the ability of TFPI-2 to inhibit plasmin, we were interest...