Abstract 2359: Cellular origin and functional contribution of alternatively spliced tissue factor to the malignant progression of chronic liver disease
Authors: Lewis Clayton S. , Stone Kateryna , Kuhnell Damaris , Langevin Scott , Soto-Gutierrez Alejandro , Van Dreden Patrick , Wang Jiang , Luyendyk James , Bogdanov Vladimir 
Published: April 2023

Background: Expression of alternatively spliced Tissue Factor (asTF), an activator of αvβ3 and α6β1 integrins, is low in the healthy liver. asTF levels in plasma exhibit a biphasic pattern in the progression of chronic liver disease (CLD): spiking in early fibrosis then declining until the onset of cirrhosis, after which asTF levels rise again in hepatocellular carcinoma (HCC). asTF can promote angiogenesis and monocyte invasion, two key features of early fibrosis and the development of HCC, the risk of which is up to 45-fold higher in cirrhotic patients. The cellular source of asTF in the liver and the mechanisms that promote its expression there are unknown. Aim: To explore the cellular origin and mechanistic underpinnings of asTF’s upregulation in CLD and HCC.

Methods: Extracellular microvesicles (MVs) and asTF protein were measured in CLD plasma. MVs were concentrated by ultracentrifugation, quantified using CountBright™ Plus Absolute Counting Beads and characterized by flow cytometry. Plasma asTF was measured by ELISA. Expression of asTF protein in liver sections of mice with experimental hepatic fibrosis (carbon tetrachloride (CCl4) or high trans-fat diet (HTFD)) and in human liver (healthy, early fibrotic, late fibrotic, cirrhotic, and HCC) was examined by immunohistochemistry. asTF mRNA and protein expression levels were studied in human hepatocyte-like HCC cell lines HepG2 and Huh7, LX-2 (stellate cells), and TMNK1 (liver sinusoidal endothelial cells). TMNK1 and LX-2 were stimulated with TNFα (20 ng/mL) and angiotensin II (10 nM).

Results: Plasma asTF levels associated positively with the levels of endothelial MVs; the level of significance rose with CLD severity. Compared to control mice, asTF staining was strong in central vein endothelium in CCl4-challenged and HTFD-fed mice. asTF co-localized with CD31 and CD68 in the human liver; again, asTF positivity rose with disease severity. In agreement with our earlier findings in human plasma, asTF expression was minimal in healthy tissue, high in early-stage fibrosis, and moderate in late-stage fibrosis; HCC tissues were highly positive for asTF. Constitutive asTF expression was low in HepG2 and Huh7 cells compared to LX-2 and TMNK1 cells. Treatment of TMNK1 cells with TNFα induced asTF protein expression, which persisted beyond 6 hours post-induction. TNFα treatment of TMNK1 cells increased the release of asTF protein and CD31+ MVs. Treatment of LX-2 cells with angiotensin II also induced transient asTF expression. Compared to control cells, asTF-overexpressing TMNK1 cells facilitated transmigration of monocytes in Boyden chamber assays.

Conclusions: Non-parenchymal cells likely contribute to the increased levels of asTF in CLD. While the precise role of asTF in progression of CLD requires further study, monocyte accumulation in liver tissue is likely to be facilitated by asTF.

Citation Format: Clayton S. Lewis, Kateryna Stone, Damaris Kuhnell, Scott Langevin, Alejandro Soto-Gutierrez, Patrick Van Dreden, Jiang Wang, James Luyendyk, Vladimir Bogdanov. Cellular origin and functional contribution of alternatively spliced tissue factor to the malignant progression of chronic liver disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2359.