A Primary Hepatic Malignant Mesenchymal Tumor with Myofibrogenic Differentiation in a B6C3F1 Mouse

Abstract

We found a malignant mesenchymal tumor with myofibrogenic differentiation in the liver of a 110-week-old female B6C3F1 mouse used for a carcinogenicity study. Sclerous white patches (maximum size: 20 × 14 mm) were observed mainly in the median lobe of the liver at necropsy. Histologically, the tumor was composed of interlacing fascicles of spindle-shaped cells with oval or elongated nuclei and lightly eosinophilic cytoplasm. Tumor cells metastasized to the lung, parapancreatic lymph node, and spleen. Special staining revealed individual tumor cells surrounded by reticulin fibers and an abundant collagenous matrix. Immunohistochemically, the tumor cells were positive for vimentin, α-smooth muscle actin, and desmin, but were negative for von Willebrand factor, Mac-2, S-100, and cytokeratin. Electron microscopic examination revealed that the tumor cells contained prominent rough endoplasmic reticulum and thin filaments in the cytoplasm, although they lacked basal lamina, focal densities, or lipid droplets. Collagenous fibers were observed in the intercellular matrix. Thus, detailed histopathological examination suggested the origin of the present tumor to possibly be Ito cells within the fibrous stroma. This report provides additional histopathological evidence of malignant hepatic nonepithelial tumors in mice.

Case Report

Hepatocellular adenomas and carcinomas are the most common neoplasms in the liver of aging mice (Harada et al. 1996). As nonepithelial tumors of the liver, hemangiomas, hemangiosarcomas, and histiocytic sarcomas (Kupffer cell sarcomas) are occasionally observed, whereas Ito cell tumors are rare in aging mice (Deschl et al. 2001; Harada et al. 1999). On the other hand, spontaneous or chemically induced hepatic sarcomas such as fibrosarcomas are very uncommon in mice (Harada et al. 1999). Only four sarcomas were identified in a search of the National Toxicology Program database of 4074 males and 4156 females, all untreated B6C3F1 mice (Harada et al. 1999). In addition to those in rodents, leiomyosarcomas and fibrosarcomas of the liver have been reported in dogs and cats (Cullen and Popp 2002; Hirao et al. 1974; Patnaik et al. 1980; Patnaik 1992). Primary hepatic malignant mesenchymal tumors such as leiomyosarcomas have also been reported in humans (Gates et al. 1995; Ishak et al. 1999). To our knowledge, however, there have been no reports on primary hepatic mesenchymal tumors such as fibrosarcoma in mice. Herein, we describe the gross, light, and electron microscopic characteristics of a hepatic malignant mesenchymal tumor with myofibrogenic differentiation in an aged mouse.

The animal was a 110-week-old female Crj:B6C3F1 (C57BL/6N × C3H/HeN) mouse in the low-dose group of a carcinogenicity study. There were no treatment-related hepatic lesions in any treated animals in this study. The animal was housed individually in an aluminum cage with a stainless steel wire mesh front and floor, under barrier conditions of 23°C ± 3°C room temperature, 55% ± 20% relative humidity, and a twelve-hour light–dark cycle. The animal had free access to a standard radiation-sterilized laboratory diet (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water via an automatic water supply system. All animal care and procedures were performed in accordance with the Guidance for Care and Use of Laboratory Animals at the Biosafety Research Center, Foods Drugs and Pesticides (An-Pyo Center). The animal was observed for clinical signs twice a day (in the morning and afternoon). When the animal showed clinical signs of emaciation and an abdominal mass at 110 weeks of age, it was euthanatized by exsanguination via the abdominal aorta. The euthanasia was performed under ether anesthesia during terminal necropsy.

At necropsy, sclerous white patches (maximum size: 20 × 14 mm) were observed, mainly in the median lobe of the liver (Figure 1). After gross postmortem examinations, systemic organs including the liver were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin (H&E) for routine histopathological examination. Then, serial sections of the hepatic lesion were stained with Masson’s trichrome stain, Watanabe’s reticulin stain, and immunohistochemically with the antibodies for vimentin (clone: V9, DAKO, Denmark, dilution 1:50, antigen retrieval by proteinase K at 37°C for ten minutes), α-smooth muscle actin (α-SMA, clone: 1A4, DAKO, dilution 1:50), desmin (clone: D33, DAKO, dilution 1:50), von Willebrand factor (rabbit polyclonal, CHEMICON International, Inc., USA, dilution 1:100), Mac-2 (clone: M3/38, CEDARLANE, Canada, dilution 1:100), S-100 (rabbit polyclonal, DAKO, dilution 1:200), tenascin-C (clone: MTn-12, Abcam, Tokyo, Japan, dilution 1:100, antigen retrieval by proteinase K at 37°C for ten minutes), cytokeratin (clone: MNF116, DAKO, dilution 1:50, antigen retrieval by proteinase K at 37°C for ten minutes), and proliferating cell nuclear antigen (PCNA, clone: PC10, DAKO, dilution 1:50) using a labeled streptavidin-biotin method (LSAB2 System-HRP, DAKO, CA, USA). Antigen retrieval for immunohistochemical staining except for anti-vimentin, anti-cytokeratin, and anti-tenascin-C was performed using a pressure cooker (DC2002 Decloaking Chamber, BIOCARE MEDICAL, CA, USA) at 125°C for five minutes and at 90°C for ten seconds in citrate buffer (pH 6.0). To confirm the specificity of antibodies, tissue sections of typical histiocytic sarcoma, hemangiosarcoma, lipomatous Ito cell tumor, the normal peripheral nerves, and gall bladders in aging mice were also prepared as positive controls for Mac-2, von Willebrand factor, tenascin-C, S-100, and cytokeratin. For electron microscopic examination, formalin-fixed hepatic tumor tissues were cut into small pieces, refixed with 2.5% glutaraldehyde and 1% osmium tetroxide, embedded in epoxy resin, sectioned, and stained with uranyl acetate and lead citrate.

Histologically, the tumor was composed of interlacing fascicles of spindle-shaped cells with oval or elongated nuclei and lightly eosinophilic cytoplasm (Figures 2a and 2b). Atrophic and deformed hepatocytes were only occasionally found intermingled among the dominant growth of tumor cells in the median lobe. In the posterior right lobe, tumor cells had apparently grown in the hepatic sinusoidal spaces (Figure 2c). Fibrous lesions were widely observed in the left lateral lobe. There were few mitotic figures in the tumor, whereas proliferative activity of the tumor cells was confirmed by PCNA staining in the posterior right lobe (Figure 2d). Lack of giant cells would suggest that the tumor was not a histiocytic sarcoma, since histiocytic sarcomas in mice often have giant cells. Likewise, absence of vascular growth suggested that the tumor was not of vascular origin. However, the tumor cells in this animal were characterized by malignant properties, showing numerous metastases to the lung, parapancreatic lymph node, and the capsule of the spleen (Figure 2e).

The stromal matrix of the tumor cells stained light blue with Masson’s trichrome stain, suggesting collagenous fibers (Figure 3a). Watanabe’s reticulin stain revealed individual tumor cells surrounded by reticulin fibers and an abundant collagenous matrix (Figure 3b). Immunohistochemically, these tumor cells were positive for vimentin, α-SMA, and desmin (Figures 3c, 3d, and 3e) but were negative for von Willebrand factor, Mac-2, S-100, and cytokeratin (data not shown). Metastatic tumor cells in the lymph node and spleen were also positive for α-SMA and desmin (Figure 2f). Unfortunately, we were not able to evaluate tenascin-C immunoreactivity in this tumor, because the tissue section from a typical Ito cell tumor of lipomatous type, used as a positive control, did not react to this antibody. Electron microscopic examination revealed that tumor cells contained prominent rough endoplasmic reticulum and thin filaments in the cytoplasm (Figure 4a). Collagenous fibers were observed in the intercellular matrix (Figure 4b), which might correlate with the Masson’s trichrome stain and Watanabe’s reticulin stain properties. No distinct characteristics of smooth muscle cells, such as basal lamina, focal densities, or pinocytotic vesicles, were detected in the tumor cells (Ghadially 1988). Ultrastructurally, the tumor cells appeared to be fibroblasts or myofibroblasts rather than smooth muscle cells. On the other hand, tumor cells had no ultrastructural features of Kupffer cells such as intracytoplasmic lysosomes.

Myofibroblast-like cells are known to play a key role in hepatic fibrogenesis (Moreira 2007). On the other hand, hepatic stellate cells (HSCs), formerly known as Ito cells, lipocytes, or perisinusoidal cells, have been noted to be the main collagen-producing cells in the liver (Friedman et al. 1985). In the normal liver, HSCs reside in the space of Disse and are the major storage sites of vitamin A. Following liver injury, HSCs become activated and transdifferentiate into myofibroblast-like cells, acquiring contractile, proinflammatory, and fibrogenic properties (Marra 1999; Milani et al. 1990). Some studies have demonstrated the presence of desmin in the cytoplasm of Ito cells in the rat liver (Yokoi et al. 1984), and proliferated Ito cells reportedly show enhanced immunostaining for desmin and actin in the experimentally injured rat liver (Ogawa et al. 1986).

Detailed histopathological examination suggested that the origin of the present tumor might be Ito cells. To our knowledge, only “benign” Ito cell tumors have been reported in aging mice (Dixon et al. 1994; Tillmann et al. 1999), whereas there have been no reports of “malignant” Ito cell tumors in mice. Reports of benign Ito cell tumors described two types of components in proliferative areas (Dixon et al. 1994; Kotani et al. 2007; Tillmann et al. 1999). One type is a lipomatous lesion composed of signet-ring-shaped cells, and the other type consists of spindle-shaped cells proliferating among disrupted hepatic plates with a stromal matrix closely resembling the tumor morphology seen in the present case. Recently, a neoplasm of HSCs (Ito cells) as a new tumor entity was reported in a human liver tumor (Kaiserling and Müller 2005). This tumor was characterized by an unusual accumulation of spindle cells surrounded by collagen matrix in the liver sinusoids, and expressed vimentin and smooth muscle actin. These histological findings were similar to those of the animal described herein. Tillmann et al. (1999) demonstrated tenascin immunoreactivity in murine Ito cell tumors. Tenascin is one of the extracellular matrix proteins, and it was demonstrated in cultured desmin-positive Ito cells by the double-label immunofluorescence technique (Van Eyken et al. 1992). Although tenascin expression is considered to be supportive evidence for the diagnosis of a murine Ito cell tumor, we were not able to demonstrate tenascin immunoreactivity in the present case.

It was assumed that this tumor, despite being found in a treated mouse, was not related to the compound being studied because of the unique occurrence of this tumor in treated animals. None of the other treated animals in this study showed any treatment-related hepatic lesions. Moreover, to date, no evidence of chemically related neoplastic effects has been reported for this compound.

Based on the results of histopathological examinations described above, we diagnosed a spontaneous malignant mesenchymal tumor with myofibrogenic differentiation, which had developed in the liver of an aged mouse. To our knowledge, this is the first report of a myofibrogenic differentiated sarcoma of the liver in a mouse, and the findings described herein provide additional histopathological evidence of malignant hepatic nonepithelial tumors in mice.