Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential

Rodrigo Carlessi; Elena Denisenko; Ebru Boslem; Julia Köhn-Gaone; Nathan Main; N Dianah B Abu Bakar; Gayatri D Shirolkar; Matthew Jones; Aaron B Beasley; Daniel Poppe; Benjamin J Dwyer; Connie Jackaman; M Christian Tjiam; Ryan Lister; Michael Karin; Jonathan A Fallowfield; Timothy J Kendall; Stuart J Forbes; Elin S Gray; John K Olynyk; George Yeoh; Alistair R R Forrest; Grant A Ramm; Mark A Febbraio; Janina E E Tirnitz-Parker

doi:10.1016/j.xgen.2023.100301

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential

Cell Genom. 2023 Apr 13;3(5):100301. doi: 10.1016/j.xgen.2023.100301. eCollection 2023 May 10.

Authors

Rodrigo Carlessi^{1

2}, Elena Denisenko², Ebru Boslem³, Julia Köhn-Gaone¹, Nathan Main¹, N Dianah B Abu Bakar¹, Gayatri D Shirolkar¹, Matthew Jones², Aaron B Beasley⁴, Daniel Poppe^{2

5}, Benjamin J Dwyer¹, Connie Jackaman¹, M Christian Tjiam^{1

6}, Ryan Lister^{2

5}, Michael Karin⁷, Jonathan A Fallowfield⁸, Timothy J Kendall^{8

9}, Stuart J Forbes¹⁰, Elin S Gray⁴, John K Olynyk⁴, George Yeoh², Alistair R R Forrest², Grant A Ramm¹¹, Mark A Febbraio³, Janina E E Tirnitz-Parker^{1

2}

Affiliations

¹ Curtin Medical School, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA 6102, Australia.
² Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, WA 6009, Australia.
³ Cellular & Molecular Metabolism Laboratory, Monash Institute of Pharmacological Sciences, Monash University, Parkville, VIC 3052, Australia.
⁴ School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA 6027, Australia.
⁵ ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Nedlands, WA 6009, Australia.
⁶ Wesfarmers Centre of Vaccines and Infectious Diseases, Telethon Kids Institute, Nedlands, WA, Australia.
⁷ Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
⁸ University of Edinburgh Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK.
⁹ Edinburgh Pathology, University of Edinburgh, Edinburgh, UK.
¹⁰ Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
¹¹ Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia.

Abstract

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Keywords: HCC; cancer surveillance; chronic liver disease; daHep; disease-associated hepatocyte; hepatocellular carcinoma; pre-malignancy; prognostic biomarker; snRNA-seq.