Molecular and immunological profiling for biomarker discovery in NAFLD-HCC

Incidence of NAFLD-HCC has increased globally but its molecular and immunological features have not been fully characterized. To search its predictive biomarkers, we performed RNA-sequence of liver tissues in 98 NAFLD patients and identified thrombospondin-2 (THBS2) significantly upregulated in NASH or advanced fibrosis patients. Serum levels of TSP-2, a secreted protein encoded by THBS2, were measured in another cohort of 213 NAFLD patients and significantly elevated in NASH or advanced fibrosis patients. Serum TSP-2 stratified NAFLD patients according to the risk of HCC development. Next, we performed multiomics profiling of tumor tissues in 113 non-viral HCC patients including 54 NAFLD-HCC patients. Unsupervised clustering of tumor transcriptomes identified a pro-tumor immune subclass characterized by T cell exhaustion signature, M2 macrophage and stromal infiltration, high PD-L1 expression, and TGF-β signaling activation. This subclass was frequently observed in NASH-HCC and characterized by intratumoral steatosis. Lipidomics-based intratumor free fatty acid profiling showed the increase in palmitic acid (PA) in this subclass. Lipid accumulation by PA supplementation in HCC cells upregulated PD-L1 and TGF-β expression in vitro. Furthermore, lipid-accumulated HCC cells promoted M2 polarization of co-cultured macrophages and upregutated TGF-β expression of co-cultured fibroblasts. In conclusion, we identified TSP-2 as a predictive biomarker of NAFLD-HCC development. Multiomics profiling also identified the pro-tumor immune subclass associated with NASH-HCC and suggested the link between intratumor steatosis and pro-tumor immune microenvironment.