Progression to fibrosis and hepatocellular carcinoma in DEN CCl4 liver mice, is associated with macrophage and striking regulatory T cells infiltration

Ananya Ajith¹Jonathan Evraerts¹Caroline Bouzin²Davide Brusa³Makram Merimi⁴Mehdi Najar⁵Françoise Smets¹Etienne Sokal¹Mustapha NAJIMI^1*

• ¹Laboratory of Paediatric Hepatology and Cell Therapy, Institut de recherche expérimentale et clinique, Faculté de pharmacie et des sciences biomédicales, Université catholique de Louvain, Brussels, Belgium
• ²Plateforme d'imagerie IREC, Institut de Recherche Expérimentale et Clinique, Faculté de Pharmacie et des Sciences Biomédicales, Université Catholique de Louvain, Brussels, Belgium
• ³CytoFlux-IREC Flow Cytometry and Cell Sorting Platform, Institut de recherche expérimentale et clinique, Faculté de pharmacie et des sciences biomédicales, Université catholique de Louvain, Brussels, Belgium
• ⁴Genetics and Immune Cell Therapy Unit, LBBES Laboratory, Faculty of Sciences, Mohamed Premier University, Oujda, Morocco
• ⁵Faculté de médecine, Université libre de Bruxelles, Brussels, Belgium

Background & AimHepatocellular carcinoma (HCC) is a classic inflammation related cancer with most cases arising from chronic liver disease (CLD). This study investigates immune dysregulation that occurs during the progression of CLD to HCC by delineating changes in immune cell composition and distribution within the liver microenvironment.MethodsMice were injected with Diethylnitrosamine (DEN) at 4 weeks of age, followed by continuous tri-weekly injections of carbon tetrachloride (CCl4) for 6 and 21 weeks to induce liver fibrosis and HCC. Naïve and Phosphate-buffered saline (PBS) corn oil treated mice were used as controls. Immune cell profiling was performed using multiplex immunofluorescence and flow cytometry analyses.ResultsThe spatial analysis of immune cell populations in HCC reveals stable leukocytes overall, with notable increases in myeloid cells, particularly infiltrating macrophages (Inf mph). Indeed, Inf mph show a progressive enrichment from control to tumor, reaching a 5-fold and 10-fold increase in the invasive margin (IM) and surrounding non-tumor tissue (NTT) regions, respectively. T lymphocytes, especially CD4+ T cells but not CD8+ T cells, significantly expand, with CD4+ cells increasing up to 10-fold in the IM and NTT regions of HCC livers. Regulatory T cells (Tregs) population exhibits an extraordinary 125-fold and 80-fold surge in the IM and NTT regions, respectively. ConclusionsThe DEN-CCl4 induced HCC mouse model replicates key immunosuppressive features of human HCC, notably increased Tregs and macrophages, which provides a robust platform for testing immunotherapies. The prominence of immune cells in the IM region underscores its importance as a critical interface modulating tumor-immune interactions, while the elevated immune presence in the NTT region reflects broader immune dysregulation associated with advanced CLD, and potentially facilitating tumor progression.