[CANCER RESEARCH 62, 6682– 6687, November 15, 2002] Essential Roles of Tumor Necrosis Factor Receptor p55 in Liver Metastasis of Intrasplenic Administration of Colon 26 Cells Hidekazu Kitakata, Yoko Nemoto-Sasaki, Yutaka Takahashi, Toshikazu Kondo, Masayoshi Mai, and Naofumi Mukaida 1 Divisions of Molecular Bioregulation [H. K., Y. N-S., N. M.] and Surgical Oncology, Cancer Research Institute [H. K., Y. T., M. M.], and Division of Environmental, Forensic, and Environmental Social Medicine, Graduate School of Medicine [T. K.], Kanazawa University, Kanazawa 920-0934, Japan ABSTRACT Intrasplenic administration of a colon adenocarcinoma cell line, colon 26, induced tumor necrosis factor (TNF)  protein expression around the central and portal veins of the liver at 3 days, and liver metastases by 24 days after the tumor injection, in 90% of wild-type (WT) mice. To explore the roles of TNF- in the process, we admin- istered colon 26 cells into tumor necrosis factor receptor p55 (TNF- Rp55) knockout (KO) mice. Less than 50% of TNF-Rp55 KO mice developed liver metastasis with significantly lower liver weights and the volumes of metastatic foci. These observations suggest the critical roles of TNF-Rp55-mediated signals in this liver metastasis model. The intrasplenic tumor injection induced mRNA expressions of vascular endothelial growth factor, heparin-binding epidermal growth factor, matrix metalloproteinase-9, and tissue inhibitor of matrix metallopro- teinase-1 at similar levels in the livers of both WT and TNF-Rp55 KO mice. Immunohistochemical analyses of the livers of WT mice after tumor injection demonstrated the enhanced expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on sinusoidal endothelial cells. Enhanced E-selectin expression was similarly observed in the liver of TNF-Rp55 KO mice after tumor injection. However, the en- hancement in VCAM-1 mRNA expression and its protein production was significantly attenuated in the liver of TNF-Rp55 KO mice when compared with WT mice. Collectively, these observations suggest that TNF-Rp55-mediated signals can up-regulate both VCAM-1 expression in the liver and subsequent liver metastasis after intrasplenic tumor injection. INTRODUCTION Despite recent advances in diagnostic and therapeutic measures, the prognosis of cancer patients with metastasis still remains poor. Thus, it is mandatory to elucidate the molecular mechanism of the metas- tasis process to develop appropriate treatment modalities. Accumu- lating evidence indicates that progressive tumor growth depends on a series of sequential and highly selective steps, each of which is rate limiting (1). The first step is tumor growth at the primary site, which is supported by neovascularization (1, 2). Subsequently, tumors de- grade basement membranes mediated by MMPs 2 and invade venules or lymphatics (3). Tumor cells then circulate, and those expressing adhesion molecules adhered to capillary beds in distant organs (4). Adherent tumor cells extravasate into tissues by degrading basement membranes with MMPs. Finally, the tumor cells proliferate in distant organs, in association with neovascularization. TNF- was originally identified as a cytokine responsible for endotoxin-induced tumor necrosis (5). Several independent groups reported that therapy with recombinant TNF- was effective against several types of murine tumor models of hepatic (6, 7) and pulmonary metastasis (8), particularly when it was administered in combination with IFN- (9) or IL-2 (10, 11). Moreover, a Phase I clinical study of TNF- treatment demonstrated partial efficacy against metastatic spread (12, 13). However, in several models, the administration of TNF- or the TNF- gene transduction into tumor cells enhanced the incidence of metastasis (14 –17). These contradictory results may be explained by differences in the cell types used in each experiment (17). Consequently, the role of endogenous TNF- in the metastatic process remains to be determined. TNF- uses two types of receptors, encoded by distinct genes: a TNF receptor with a molecular weight of M r 75,000 (TNF-Rp75) and one with a molecular weight of M r 55,000 (TNF-Rp55; Ref. 18). These two receptors show 30% homology at the amino acid level in their extracellular, cysteine-rich, and ligand-binding regions (19). TNF-Rp75 expression is restricted to hematopoietic and endothelial cells, and mediates a limited number of the biological activities of TNF-, including thymocyte and T-cell proliferation (20). In contrast, TNF-Rp55 is ubiquitously expressed on almost all cell types except for RBCs, and mediates the various activities of TNF-. Therefore, we investigated the role of TNF- in the spread of tumor metastases. Prior studies have revealed that p55 but not p75 is ex- pressed abundantly in liver. 3 Hence, to evaluate the roles of TNF-, particularly TNF-Rp55-mediated signals, we injected a murine colon adenocarcinoma cell line into the spleen of WT and TNF-Rp55 KO mice. This approach has provided definitive evidence that the absence of TNF-Rp55 results in attenuated VCAM-1 expression in the liver and also reduced the incidence of liver metastasis. MATERIALS AND METHODS Mice. Specific pathogen-free 8 –12-week old female BALB/c mice were purchased from Charles River Japan Co. (Yokohama, Japan) and designated as WT mice. TNF-Rp55-deficient mice were a kind gift of Dr. Horst Bluethmann (Hoffmann-Roche, Basel, Switzerland; Ref. 21), and were backcrossed to BALB/c mice for 6 generations in our animal facility (22) and designated as TNF-Rp55 KO mice. All of the animal experiments were performed in accordance with the Guideline for the Care and Use of Laboratory Animals in the Takara-machi Campus of Kanazawa University. Induction of Liver Metastasis. A murine adenocarcinoma cell line, colon 26 (23), was maintained in DMEM with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere with 5% CO 2 . Subcon- fluent cells were collected and resuspended in HBSS at a cell density of 5  10 5 cells/ml. The cell viability was always 95% by a trypan blue exclusion test. One-hundred l of cell suspension was injected into the spleens of mice under light ether anesthesia. Mice were sacrificed at the indicated time intervals after tumor injections to determine the incidence of liver metastasis, the number of metastatic foci in the liver, and the tumor size. Tumor size was calculated according to an equation of a  b 2 /2, where a and b indicate long and short diameters of the tumor, respectively (24). In another series of experiments, the livers were removed from the mice at Received 4/4/02; accepted 9/20/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. Phone: 81-76-265-2767; Fax: 81-76-234-4520; E-mail: naofumim@kenroku.kanazawa-u.ac.jp. 2 The abbreviations used are: MMP, matrix metalloproteinase; TNF, tumor necrosis factor; IL, interleukin; TNF-R, tumor necrosis factor receptor; VCAM-1, vascular cell adhesion molecule-1; WT, wild-type; KO, knockout; ICAM-1, intercellular adhesion molecule-1; RT-PCR, reverse transcription-PCR; TIMP, tissue inhibitor of matrix metal- loproteinase; VLA-4, vary late antigen-4. 3 T. Kondo and N. Mukaida, unpublished observations. 6682 Research. on November 24, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from