Spatial transcriptomics supports a role for SOX-4 driven signaling throughout the disease course of biliary atresia

Biliary atresia (BA) is a fibroinflammatory cholangiopathy of infancy that is the most common indication for pediatric liver transplantation. We aimed to define the molecular mechanisms responsible for differences in the rate of disease progression among children with BA. We performed spatial transcriptomics (ST) analysis on frozen liver tissue at transplant from 16 children: BA with survival with native liver (SNL) <2 years (BA1, n=3, BA1_3 was excluded from analysis), BA with SNL >2 years (BA2, n=4), non-BA cholestasis (n=4), and non-diseased donors (n=4). Transcriptional signatures were compared between patient groups by tissue region (scar, hepatocyte, cholangiocyte). Findings were validated in larger patient cohorts that included BA samples at diagnosis. ST analysis of BA1 patients showed the most aggressive disease phenotype, characterized by reduced hepatocyte zonation, low expression of homeostatic metabolic signatures, and increased scar heterogeneity enriched for pathways including extracellular matrix remodeling, interferon response, and leukocyte activation. Notably, genes involved in SOX4 hepatocyte-to-cholangiocyte reprogramming were most enriched in BA1 patients. Liver immunohistochemistry with in situ mRNA hybridization showed that BA patients at diagnosis had increased SOX4 quantification as compared to BA patients at transplant. Lastly, previously published liver bulk RNA-sequencing data demonstrated higher SOX4 gene-set expression in BA patients at diagnosis with SNL < 2 years. Children with BA and worse outcome exhibit increased SOX4 gene-set expression at diagnosis with greater loss of hepatocyte zonation and immune-driven scar heterogeneity at transplant. Further mechanistic studies are needed to determine whether SOX4-driven biliary reprogramming plays a maladaptive role in the reparative response to obstructive cholestasis in BA.