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Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.

NK4 suppresses invasion and metastasis of tumor cells by means of dual actions as HGF antagonist and angiogenesis inhibitor. Our previous studies showed that NK4 suppresses the implantation of tumor cells to the peritoneal milky spots (MS) by intraperitoneal injection (i.p.) of adenovirus vector expressing NK4 (Ad-NK4) or NK4 gene-transfected tumor cells. In the present study, we investigated the antitumor mechanisms of NK4 in the suppression of peritoneal implantation. When evaluated by a fluorescent microscopy, a prior injection of Ad-NK4 suppressed peritoneal implantation immediately after the injection of GFP-expressing tumor cells. DNA microarray analyses also demonstrated a reduced expression of some adhesion molecules in NK4 gene-transfected tumor cells as compared to neomycin gene-trasfected cells (control). In the in vitro adhesion assay, the adhesion to some types of the extra cellular matrixs (ECM) was significantly decreased in NK4 gene-transfected cells as compared to the control. These results suggest that NK4 may suppress peritoneal implantation by inhibiting adhesion of tumor cells to ECM around MS.

Tumor-stromal interactions, which are regulated by stromal-derived HGF and tumor-derived HGF inducers, are essential for tumor cell acquisition of such malignant properties as invasion and metastasis. NK4, a proteolytic cleavage product of HGF, has antitumor activities as both an HGF antagonist and an angiogenesis inhibitor. In this study, we examined the in vitro and in vivo behaviors of mouse colon adenocarcinoma C T26 cells modified by gene transfer to secrete NK4, and investigated the influence of NK4 on expression of HGF and HGF inducers associated with tumor-stromal interactions. In vitro cell proliferation rates of NK4 transfectant (C T26-NK4) and mock transfectant (C T26-NEO) were essentially the same, and scattering and invasion were stimulated by HGF in C T26-NEO, but not in C T26-NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of C T26-NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased microvessels and increased apoptotic cells in C T26-NK4 tumor compared with control. Interestingly, HGF, strongly expressed in C T26-NEO tumor stroma, was reduced in C T26-NK4. In vitro, conditioned medium of C T26-NK4 inhibited fibroblast-derived HGF production, which was increased by that of C T26-NEO. Moreover, although similar constitutive expression levels of PDGF and TGF-alpha (both HGF inducers) were detected in C T26-NK4 and C T26-NEO in semiquantitative RT-PCR analyses, the expression was up-regulated by HGF in C T26-NEO, but not C T26-NK4. These results suggest that NK4 may exert antitumor activities not only by antagonizing HGF, but also by inhibiting HGF amplification via tumor-stromal interactions. Continuous, abundant NK4 production induced at a tumor site by gene transfer should show multiple antitumor activities with potential therapeutic benefit.

An HGF antagonist, NK4, inhibits not only invasion and metastasis of tumor cells driven by HGF-Met receptor binding, but also tumor angiogenesis. To address the antitumor activities of NK4, we investigated the biological behaviors of CT26 transfected with the NK4 gene (CT26-NK4) in vitro and in vivo. In the in vitro assay, the invasion in MOCK transfected cells (control) was stimulated by HGF; however, in CT26-NK4 cells, these effects were completely inhibited. In the in vivo assay, the tumor growth of CT26-NK4 was strongly suppressed and the survival of CT26-NK4 tumor-bearing mice was significantly prolonged. Immunohistochemical analysis revealed that while proliferating cells (PCNA immunostaining) of CT26-NK4 tumors were weakly suppressed, the micro-vessel number (CD31/PECAM-1 immunostaining) in those tumors was significantly suppressed as compared with the control tumors. In conclusion, NK4 exerts potent antitumor effects via anti-angiogenesis rather than inhibition of biological events of tumor cells stimulated by HGF.