Spatial proteomics of immune microenvironment in nonalcoholic steatohepatitis-associated hepatocellular carcinoma

Background and Aims: NASH-HCC is inherently resistant to immune checkpoint blockade, but its tumor immune microenvironment is largely unknown. Approach and Results: We applied the imagingmass cytometry to construct a spatially resolved single-cell atlas from the formalin-fixed and paraffin-embedded tissue sections from patients with NASH-HCC, virus-HCC (HBV-HCC and HCV-HCC), and healthy donors. Based on 35 biomarkers, over 750,000 individual cells were categorized into 13 distinct cell types, together with the expression of key immune functional markers. Higher infiltration of T cells, myeloid-derived suppressor cell (MDSCs), and tumor-associated macrophages (TAMs) in HCC compared to controls. The distribution of immune cells in NASH-HCC is spatially heterogeneous, enriched at adjacent normal tissues and declined toward tumors. Cell-cell connections analysis revealed the interplay of MDSCs and TAMs with CD8(+) T cells in NASH-HCC. In particular, exhausted programmed cell death 1 (PD-1(+))CD8(+) T cells connected with programmed cell death-ligand 1 (PD-L1(+))/inducible T cell costimulator (ICOS+) MDSCs and TAMs in NASH-HCC, but not in viral HCC. In contrast, CD4(+)/CD8(+) T cells with granzyme B positivity were reduced in NASH-HCC. Tumor cells expressed low PD-L1 and showed few connections with immune cells. Conclusions: Our work provides the first detailed spatial map of single-cell phenotypes and multicellular connections in NASH-HCC. We demonstrate that interactions between MDSCs and TAMs with effector T cells underlie immunosuppression in NASH-HCC and are an actionable target.