Abstract
Transforming growth factor (TGF)-β stimulates extracellular matrix (ECM) deposition during development of liver fibrosis and cirrhosis, the most important risk factor for the onset of hepatocellular carcinoma. In liver cancer, TGF-β is responsible for a more aggressive and invasive phenotype, orchestrating remodeling of the tumor microenvironment and triggering epithelial–mesenchymal transition of cancer cells. This is the scientific rationale for targeting the TGF-β pathway via a small molecule, galunisertib (intracellular inhibitor of ALK5) in clinical trials to treat liver cancer patients at an advanced disease stage. In this study, the hypothesis that galunisertib modifies the tissue microenvironment via inhibition of the TGF-β pathway is tested in an experimental preclinical model. At the age of 6 months, Abcb4ko mice—a well-established model for chronic liver disease development and progression—are treated twice daily with galunisertib (150 mg/kg) via oral gavage for 14 consecutive days. Two days after the last treatment, blood plasma and livers are harvested for further assessment, including fibrosis scoring and ECM components. The reduction of Smad2 phosphorylation in both parenchymal and non-parenchymal liver cells following galunisertib administration confirms the treatment effectiveness. Damage-related galunisertib does not change cell proliferation, macrophage numbers and leucocyte recruitment. Furthermore, no clear impact on the amount of fibrosis is evident, as documented by PicroSirius red and Gomori-trichome scoring. On the other hand, several fibrogenic genes, e.g., collagens (Col1α1 and Col1α2), Tgf-β1 and Timp1, mRNA levels are significantly downregulated by galunisertib administration when compared to controls. Most interestingly, ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic β-catenin pathway, are remarkably reduced by galunisertib-treated Abcb5ko mice. In conclusion, TGF-β inhibition by galunisertib interferes, to some extent, with chronic liver progression, not by reducing the stage of liver fibrosis as measured by different scoring systems, but rather by modulating the biochemical composition of the deposited ECM, likely affecting the fate of non-parenchymal cells.
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Acknowledgements
We thank Christof Dormann and Friedrich Behne for their excellent technical support.
Funding
This work was supported by the BMBF program LiSyM (SD: Grant PTJ-FKZ: 031 L0043), e:Bio-Modull-II: MS_DILI and by the Italian Ministry of Health, Ricerca Corrente 2018.
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SH, JW and AD conceived the study, performed data analyses and wrote the manuscript. EC, MLC and AI performed the pathological evaluation. MPE, SD and GG performed critical revision of the manuscript. SD and GG provided supervisory support and corrected the manuscript. All authors read the final version of the manuscript.
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204_2018_2231_MOESM1_ESM.pptx
Fig. S1 Liver cell proliferation, resident macrophage and leucocyte numbers upon Galunisertib treatment in 26-weeks-old Abcb4ko mice. a) Representative images of Ki-67 (proliferation marker), F4/80 (resident macrophages) and CD45 (leukocyte common marker) positive signals in liver tissues upon Galunisertib or vehicle administration. Scale bars are 100 µm. b and c) show numbers of proliferating hepatocytes and non-parenchymal cells, as indicated by Ki-67 positivity. d and e) quantification of F4/80 positive areas and number of CD45 positive cells, among different groups. Data are means ± SD of 5–7 mice per group. N.S.: P > 0.05; **: P < 0.01 compared with untreated livers unless otherwise indicated. (PPTX 4035 KB)
204_2018_2231_MOESM2_ESM.pptx
Body and liver weight as well as liver enzymes upon Galunisertib treatment in 26-weeks-old Abcb4ko mice. a–c mouse and liver weight, and liver as percentage of total body weight. No significant difference is present due to Galunisertib administration. d–f) liver enzymes in Galunisertib, vehicle and untreated mice. e) H&E staining across different groups. Scale bars are 200 µm. Data are means ± SD of 5–7 mice per group. N.S.: P > 0.05; *: P < 0.05; **: P < 0.01 compared with untreated livers unless otherwise indicated. (PPTX 1695 KB)
204_2018_2231_MOESM3_ESM.pptx
Negative controls for IHC as used in this study. In the same staining batch, negative controls by omitting first antibodies were included. Scale bars are 100 µm. (PPTX 5991 KB)
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Abcb4ko mouse age, gender and number. Experiment 1 was used to investigate chronic liver disease development and progression. Experiment 2 was performed to test the anti-fibrotic efficiency of Galunisertib. (PPTX 36 KB)
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PBC scoring of liver fibrosis in 12 to 84 weeks old Abcb4ko mice. Correlation analysis is based on Test z versus 12-weeks-old mice (PPTX 69 KB)
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Hammad, S., Cavalcanti, E., Werle, J. et al. Galunisertib modifies the liver fibrotic composition in the Abcb4Ko mouse model. Arch Toxicol 92, 2297–2309 (2018). https://doi.org/10.1007/s00204-018-2231-y
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DOI: https://doi.org/10.1007/s00204-018-2231-y