HEPATOCELLULAR CARCINOMA IN AN ORTHOTOPIC MOUSE MODEL METASTASIZES INTRAHEPATICALLY IN CIRRHOTIC BUT NOT IN NORMAL LIVER Shigeki KURIYAMA*, Masaharu Y AMAZAKI, Akira MITORO, Tatsuhiro T SUJIMOTO, Masaji KIKUKAWA, Hirohisa T SUJINOUE, Toshiya NAKATANI, Yasunori T OYOKAWA, Hitoshi Y OSHIJI and Hiroshi FUKUI Third Department of Internal Medicine, Nara Medical University, Nara, Japan Prognosis of hepatocellular carcinoma (H CC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of H CC in cirrhotic liver. T o examine this, a liver cirrhosismodel wasproduced by injecting thioacetamide i.p. into mice. Murine H CC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal liversof syngeneic mice. DiI-labeled H CC cells in the liver were observed under fluorescent and confocal microscopy. H istological analysis of cirrhotic and normal livers revealed that implanted H CC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. T his characteristic behavior of H CC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cir- rhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. T hus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of H CC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of varioustypesof cancer. Int. J. Cancer 80:471–476, 1999.  1999 Wiley-Liss, Inc. Although primary cancers of the liver include multiple histologic sub-types, hepatocellular carcinoma (HCC) accounts for more than 90% of primary hepatic malignancies. The incidence of HCC is quite variable worldwide, related to variable distribution of predisposing factors. In low-incidence areas, such as the United States and Western Europe, HCC has an incidence of 3 to 7 cases per 100,000 population. HCC is much more frequently seen in areas of sub-Saharan Africa and Southeast Asia, where the reported incidence approaches 150 cases per 100,000 (Simonetti et al., 1991). In Japan, approximately 85% of malignant neoplasms in the liver are assumed to be hepatocellular carcinoma. The death rate from HCC has gradually increased both for males and for females during the past 3 decades. HCC is responsible for approxi- mately 27,000 fatalities a year, and represents the third and fourth most frequent causes of malignant-neoplasm-related deaths in males and females, respectively (Hatori, 1994). Any patient with chronic liver disease is at increased risk for the development of HCC. Predisposing factors for HCC include cirrhosis, chronic hepatitis and various carcinogens, and a minority of HCCs are thought to arise de novo in non-cirrhotic livers. In the majority of cases (70–90%), HCC is found in conjunction with liver cirrhosis. HCC is known to have a poor prognosis because of the high frequency of multiple hepatic tumors, although patients rarely have distant metastases at the time of surgery. Although some cases of HCC have a multicentric origin, multiple lesions in HCC have been attributed mainly to intrahe- patic metastases (Okuda and Nakashima, 1981). Intrahepatic metastases were found in approximately 30% of cases of surgically resected small HCC with a diameter of 3 cm and in almost 80% of HCC autopsy cases (Yuki et al., 1990). Early and liver-specific recurrences have also been seen frequently in cases of HCC treated by liver transplantation (McPeake et al., 1993). These findings indicate that the liver is the main target organ of HCC metastasis. Although it has been suggested, from clinical observation, that intrahepatic metastasis may be generated by cell implantation via the portal venous system (Nakashima, 1976), the mechanism and pathogenesis of the high frequency of intrahepatic metastasis and portal invasion in HCC are not well understood. To investigate the mechanism of intrahepatic invasion of HCC, we employed a fluorescent-dye-labeling technique. We have shown that when murine HCC cells labeled with the fluorescent carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), were implanted into the liver of syngeneic mice, HCC cells preferen- tially migrated to and invaded the adjacent periportal regions, known as Rappaport’s Zone 1, but not the adjacent centrolobular areas, Zone 3 (Kuriyama et al., 1998). DiI enters hydrophobic compartments and is stable, non-metabolized and non-diffusible, not only in vitro but also in vivo (Ledley et al., 1992). This method enables us to distinguish a single HCC cell in the liver, conse- quently it is possible to trace the early invasive and metastatic footprints of HCC in the liver. In the present study, to investigate the invasive and metastatic behavior of HCC in cirrhotic liver, we implanted DiI-labeled murine HCC cells under the capsule of the left-lateral hepatic lobe of cirrhotic mice produced by i.p. administration of thioacetamide (TAA). We investigated whether HCC preferentially invades adjacent Zone 1 but not adjacent Zone 3 in cirrhotic liver; also, we examined whether HCC metastasizes to unimplanted hepatic lobes in cirrhotic liver. MATERIAL AND METHODS Cell culture The murine HCC cell line BNL1ME A.7R.1, which was originally established from a BALB/c mouse, was purchased from the ATCC (Rockville, MD). Cells were cultured in RPMI-1640 medium supple- mented with 10% (vol/vol) heat-inactivated FCS, 0.3 mg/ml L-glutamine, 100 units/ml ampicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 in air. Labeling of cells DiI was purchased from Molecular Probes (Eugene, OR) and prepared as a 2.0 mg/ml solution in 99.5% ethanol. The DiI solution was then passed through a 0.22-μm-pore filter (Millipore, Bedford, MA), and routinely sonicated prior to application to cells. Optimal conditions for labeling murine HCC cells and hepatocytes have been described (Kuriyama et al., 1998). Briefly, cells cultured at 37°C were trypsinized and suspended in culture medium without serum at a concentration of 5  10 6 cells/ml, and stained by incubating with 20 μg/ml DiI for 60 min at 37°C with gentle agitation at 10-min intervals. After staining, the cells were washed 3 times by centrifugation to remove the dye not incorporated in the cells and re-suspended in the culture medium or in PBS. Cell viability was assessed by Trypan-blue exclusion. Grant sponsor: Japanese Ministry of Education, Science, Sports and Culture; Grant numbers: B-07457141 and B-10470140. *Correspondence to: Third Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Fax: (81) 744-24-7122. Received 25 June 1998; Revised 14 August 1998 Int. J. Cancer: 80, 471–476 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer