Satoshi Inada

Reduced HGF expression in subcutaneous CT26 tumor genetically modified to secrete NK4 and its possible relation with antitumor effects

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 anti‐tumor 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 CT26 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 (CT26‐NK4) and mock transfectant (CT26‐NEO) were essentially the same, and scattering and invasion were stimulated by HGF in CT26‐NEO, but not in CT26‐NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of CT26‐NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased micro vessels and increased apoptotic cells in CT26‐NK4 tumor compared with control. Interestingly, HGF, strongly expressed in CT26‐NEO tumor stroma, was reduced in CT26‐NK4. In vitro, conditioned medium of CT26‐NK4 inhibited fibroblast‐derived HGF production, which was increased by that of CT26‐NEO. Moreover, although similar constitutive expression levels of PDGF and TGF‐α (both HGF inducers) were detected in CT26‐NK4 and CT26‐NEO in semiquantitative RT‐PCR analyses, the expression was up‐regulated by HGF in CT26‐NEO, but not CT26‐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.

Suppression of peritoneal implantation of gastric cancer cells by adenovirus vector-mediated NK4 expression

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.