Digital spatial profiling reveals immune heterogeneity across tumor and stromal compartments in hepatocellular carcinoma

Introduction : Hepatocellular Carcinoma (HCC) is one of the most devastating malignancies around the world, especially in East Asia. Despite significant advances in immune checkpoint inhibitors in the past decade, many patients show poor response, highlighting the need to better understand tumor heterogeneity, especially at the spatial level. GeoMx™ Digital Spatial Profiler (DSP) is a spatial biology platform that enables high-plex, in situ profiling of FFPE samples, allowing molecular characterization within distinct tissue compartments while preserving spatial context. In this study, DSP was exploited for immune profiling utilizing FFPE samples from HCC patients. Methods: A total of 4 immune-related panels were used for DSP protein profiling. Thirty HCC FFPE slides were used, and 341 regions of interest were identified from tumor and tumor stroma tissue for DSP analysis. Four morphology markers (CD45/Vimentin/CK8/18/SYTO83) were selected for Immunofluorescence (IF) staining to guide the regions of interest (ROI) selection for tissue structure and distinct cell types. Results: DSP analysis revealed distinct immune patterns within HCC tissues. Principal component analysis of immune-related markers stratified regions of samples distinctly by tumor and stroma, highlighting the spatial heterogeneity of immune engagement. Notably, stromal compartments exhibited stronger differential expression of immune activation markers (e.g, CD45), particularly T cell-associated proteins such as CD3, CD4, and CD8 compared to tumor regions. Quantitative analysis demonstrated a strong correlation of CD45 expression measured by IF and DSP regardless of tissue compartments. IF staining confirmed low CD45 expression (<2%) within tumor regions in majority of HCC samples, indicating limited immune cell infiltration. However, a subset of samples still showed relatively higher CD45 expression in either the stroma, tumor, or both compartments, suggesting variable immune engagement across patients. Collectively, these results illustrate diverse patterns of immune infiltration in HCC. Conclusions: This study highlights the spatial heterogeneity of immune cell infiltration within the tumor microenvironment of HCC. Stratification of samples by tissue origin and the distinct expression of immune activation markers in stromal regions underscore the importance of compartment-specific analysis. The strong concordance between DSP and IF quantification for CD45 expressions supports the robustness of spatial profiling approaches. These findings may inform the development of spatially guided immunotherapeutic strategies.