ArticleLife & Medical SciencesInflammatory Models Drastically Alter Tumor Growth and the Immune Microenvironment in Hepatocellular Carcinoma
Introduction
Liver cancer is the fifth most common cancer in men, seventh most common cancer in women, and the third most deadly cancer worldwide 1., 2.. Liver cancer, particularly hepatocellular carcinoma (HCC), almost always develops on the background of a chronically diseased liver tissue, induced by long-term exposure to an inflammatory stimulus, such as hepatitis viral infections, excessive alcohol consumption, or metabolic syndrome 1., 3.. These stimuli lead to the induction of hepatocyte death and compensatory proliferation, hepatic stellate cell activation, and immune infiltration [3]. This process frequently results in a fibrotic and ultimately cirrhotic liver microenvironment; in this context, the cycling hepatocytes accumulate genetic alterations and eventually undergo malignant transformation [3]. This is in stark contrast with many other cancer types, in which the tissue from which the tumor arises is generally quite normal and functional 4., 5.. Indeed, 80%–90% of patients, whom have developed liver cancer after a long history of liver illness, have cirrhosis of the liver or late-stage fibrosis [2]. The fibrotic liver, with its characteristic scarring of the tissue, comes with a concurrent production of numerous cytokines, chemokines, and growth factors, all of which shape the microenvironment and its constituents during hepatocarcinogenesis and progression 6., 7., 8., 9.. With this common theme of inflammation and fibrosis accompanying and driving malignant transformation, a better understanding of the microenvironment and its effects on tumor progression can yield valuable insights into these processes as well as produce potential targets for therapeutic intervention.
To investigate the development and progression of HCC, mouse models are essential tools to dissect the complex roles of tumor cells and stromal components in the tumor microenvironment. Although numerous mouse models have been created in which liver cancer is induced either by carcinogens or by transgenic activation of oncogenes combined with inactivated tumor suppressor genes, it has been difficult to mimic the pathological process of the most common HCC types, those initiated by hepatitis viral infections. Thus, orthotopic transplantation of cancer cells, via direct injection into the liver or inoculation into the spleen that drains into the liver, has been a very valuable experimental approach for evaluating both primary tumor growth and metastatic potential 10., 11., 12.. However, in most experimental settings, the tumor cells have been implanted into a healthy liver in the animal host, a tissue background hardly resembling those in patients where HCC emerges and progresses, consequently leading to the generation of incomplete or even misleading information on the mechanistic aspects of the pathological process. In this regard, several studies have shown that manipulation of the fibrotic process could result in dramatic effects on tumorigenesis; however, they have primarily focused on the hepatic stellate cells themselves, with limited probes into the accompanying immune changes in the experimental systems 10., 13., 14., 15..
In this study, we addressed the question of whether common fibrotic stimuli could result in alterations to tumor growth in the context of orthotopic implantation models, with accompanying changes in both the immune components and tumor responses to the fibrotic tumor microenvironment. Our findings reveal a markedly different tumor microenvironment and tumor growth kinetics in a clinically relevant fibrotic context, emphasizing the necessity for more accurate modeling that takes into account the drastic changes in the tissue caused by chronic liver disease.
Section snippets
Mice
All experimental procedures described here were approved by the Duke University Animal Care and Use Committee. C57BL/6 breeding mice were purchased from The Jackson Laboratory.
Fibrosis induction
For the carbon tetrachloride model of liver fibrosis, 6- to 7-week-old male C57BL/6 mice were treated with either carbon tetrachloride diluted 1:20 in olive oil or olive oil alone at a volume of 600 µL kg−1. Mice received biweekly intraperitoneal injections for 6 weeks and were sacrificed 2 days following the 12th
Implantation of cells into a fibrotic liver results in enhanced tumor growth
To directly assess the impact of fibrosis and its concurrent inflammation, we utilized two of the most common models for the induction of fibrosis in the liver: carbon tetrachloride (CCl4) administration and BDL 6., 9.. CCl4 administration results in hepatocyte death upon its metabolism to trichloromethyl radicals (CCl3), resulting in centrilobular necrosis, inflammation, and hepatic stellate cell activation 6., 8., 9.. BDL, on the other hand, leads to cholestatic liver injury and a biliary
Discussion and conclusion
Liver cancer is a devastating disease, the progression and prognosis of which are intricately intertwined with the chronically inflamed and diseased tissue in which it develops. This crucial link has been made especially clear by previous studies showing that patterns of gene expression in diseased non-tumor liver tissue of patients with liver cancer is more predictive of survival than tumor tissue [27] and that modulating the fibrosis burden in patients modulates tumor development and
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors thank members of Anna Mae Diehl’s laboratory for technical help and discussions, members of the Duke Cancer Institute Flow Cytometry Shared Resource for their help in sorting cells, and members of Xiao-Fan Wang’s lab for helpful discussions. This work was supported by Grant CA154151, awarded to Xiao-Fan Wang by the National Cancer Institute of the United States of America.
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