Abstract
Simple SummaryHepatocellular carcinoma is the most frequent form of primary liver cancer, characterized by increasing incidence and high mortality. Animal models of hepatocellular carcinoma are widely used to study the biology of cancer and to test potential therapies. Herein, we describe how the rat model of DEN-induced hepatocellular carcinoma mimics the pathogenesis of hepatocellular carcinoma seen in humans, including liver damage, chronic inflammation, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and modulations of the liver’s immune microenvironment. Our results should help the hepatocellular carcinoma field to better tailor the use of the DEN-induced rat liver cancer model for testing specific experimental hypotheses or to perform preclinical testing. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease’s progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver’s inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.
Highlights
Liver cancer is currently the third most common cause of cancer-related death worldwide [1], with hepatocellular carcinoma (HCC) accounting for the majority of these cases.Almost all Hepatocellular carcinoma (HCC) cases are associated with liver fibrosis or cirrhosis developed from chronic liver injuries
The qPCR analyses of Collagen-1, alpha smooth muscle actin (α-SMA), transforming growth factor beta (TGF-β) and a tissue inhibitor of metalloproteinases (TIMP-1) confirmed the development and progression of liver fibrosis/cirrhosis as a result chronic diethyl nitrosamine (DEN) treatment, which was associated with a non-significant decrease in matrix metalloproteinase (MMP)2 and MMP9 expression (Figure 2b)
Since human HCC is almost universally linked with chronic inflammation and fibrosis, it is not surprising that these DEN-induced mouse models result in tumors that are distinct from human HCC [26,27], recent analyses suggest similarities between the mouse DEN-induced HCC and alcohol-induced
Summary
Almost all HCC cases are associated with liver fibrosis or cirrhosis developed from chronic liver injuries. Each underlying condition might involve different carcinogenic pathways, fibrosis and cirrhosis are regarded as crucial factors in the carcinogenesis of human liver tissue. The immune system of the liver contributes to the severity of the necrotic-inflammatory tissue damages, the establishment of fibrosis and the disease’s progression towards HCC [2]. The diethyl nitrosamine (DEN) chronically induced rat model reproduces human liver fibrosis and cirrhosis leading to HCC development [5,6], but limited information exists about pathway alterations, the involvement of inflammation, or the immune system characteristics of this model during progression to carcinoma. We thoroughly characterized DEN-induced HCC rat models during the progression and development of fibrosis and HCC, with a special focus on tumor immune microenvironments
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