UNASSIGNED: The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Individuals with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in individuals with NASH. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice.
UNASSIGNED: Here, we explored whether STAT3 activation in hepatocyte and non-hepatocyte areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD livers.Results: pSTAT3 scores in non-hepatocyte areas progressively increased with fibrosis severity (r = 0.53, p <0.001). Correlation analyses between pSTAT3 scores and expression of 1,540 immune- and cancer-associated genes revealed a large effect of STAT3 activation on gene expression changes in non-hepatocyte areas and confirmed a major role for STAT3 activation in fibrogenesis. Digital spatial transcriptomic profiling was also performed on 13 regions selected in hepatocyte and non-hepatocyte areas from four NAFLD liver biopsies with advanced fibrosis, using a customized panel of markers including pSTAT3, PanCK+CK8/18, and CD45. The regions were further segmented based on positive or negative pSTAT3 staining. Cell deconvolution analysis revealed that activated STAT3 was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells. Regression of liver fibrosis upon STAT3 inhibition in mice with NASH resulted in a reduction of HPCs, demonstrating a direct role for STAT3 in HPC expansion.
UNASSIGNED: Increased understanding of the spatial dependence of STAT3 signaling in NASH and liver fibrosis progression could lead to novel targeted treatment approaches.
UNASSIGNED: Advanced liver fibrosis is the main determinant of mortality in patients with NASH. This study showed using liver biopsies from 133 patients with NAFLD, that STAT3 activation in non-hepatocyte areas is strongly associated with fibrosis severity, inflammation, and progression to NASH. STAT3 activation was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells (SECs), as determined by innovative technologies interrogating the spatial distribution of pSTAT3. Finally, STAT3 inhibition in mice resulted in reduced liver fibrosis and depletion of HPCs, suggesting that STAT3 activation in HPCs contributes to their expansion and fibrogenesis in NAFLD.

Over the past decade, our understanding of human diseases has rapidly grown from the rise of single-cell spatial biology. While conventional tissue imaging has focused on visualizing morphological features, the development of multiplex tissue imaging from fluorescence-based methods to DNA- and mass cytometry-based methods has allowed visualization of over 60 markers on a single tissue section. The advancement of spatial biology with a single-cell resolution has enabled the visualization of cell-cell interactions and the tissue microenvironment, a crucial part to understanding the mechanisms underlying pathogenesis. Alongside the development of extensive marker panels which can distinguish distinct cell phenotypes, multiplex tissue imaging has facilitated the analysis of high dimensional data to identify novel biomarkers and therapeutic targets, while considering the spatial context of the cellular environment. This mini-review provides an overview of the recent advancements in multiplex imaging technologies and examines how these methods have been used in exploring pathogenesis and biomarker discovery in cancer, autoimmune and infectious diseases.