Spatially resolved multiplex protein profiling reveals DNA methylation–dependent microenvironmental remodeling in liver fibrosis

Liver fibrosis is a significant health concern that affects ∼300 million people globally, characterized by the excessive accumulation of extracellular matrix (ECM) components in the liver. A major contributor to liver fibrosis is fatty liver disease, which can progress to steatohepatitis when the accumulation of fat in the liver causes inflammation, cell death, and scarring. Long-standing steatohepatitis leads to liver fibrosis as scar tissue builds up and replaces healthy liver tissue, potentially progressing to life-threatening conditions, such as cirrhosis, liver failure, or hepatocellular carcinoma. DNA methylation plays a critical role in the progression of fatty liver disease and liver fibrosis by altering gene expression without modifying the DNA sequence. The integration of spatial analysis with protein profiling enhances our ability to explore the spatial organization of cellular interactions and protein expression in liver diseases, fostering a deeper understanding of the disease mechanisms. Multiplex immunofluorescence (mIF) imaging was performed to understand the spatial organization of 10 molecular targets and the cellular interaction between them across four distinct liver tissue types: wild-type (WT) regular, WT high-fat, fibrosis regular, and fibrosis high-fat. Notably, fibrotic high-fat samples displayed increased pan-cytokeratin and vascular cell adhesion molecule-1 (VCAM-1) expression, suggesting diet-aggravated injury and inflammation. Our findings highlight the interplay between epigenetic regulation, ECM remodeling, and cellular crosstalk in liver fibrosis. The spatial profiling approach provides insights into microenvironmental changes, revealing how DNA methylation influences protein localization and fibrotic progression. These results underscore the potential of spatial omics in elucidating disease mechanisms and guiding targeted therapies for metabolic liver disorders.