Extended Data Fig. 5: Spatial transcriptomic analyses revealed enriched activation and migration of SPON2⁺ NK cells.

a, UMAP embedding of all cells from donors with T cells, B cells, NK cells, and Myeloid projected. The UMAP plot shows annotations and colors for major immune cell types in the HCC ecosystem based on single-cell RNA sequencing data. b, UMAP embedding of all spatial spots from donors and relative gene marker expressions. The UMAP plot showcases the expression levels of SPON2, projected onto the same immune cell type map as shown in a, using single-cell RNA sequencing data. c, Differential expression of effector molecules in tumor-infiltrating SPON2⁺ and SPON2⁻ NK cells from HCC patients. Analysis of single-cell RNA sequencing data from HCC tumor samples (n = 15 patients) comparing the expression levels of IFNG (encoding IFN-γ, P = 8.0 × 10⁻⁴) and PRF1 (encoding Perforin, P = 8.0 × 10⁻³) between SPON2⁺ NK cells (n = 15 patients) and SPON2⁻ NK cells (n = 15 patients). Boxplots show the distribution of expression levels, with individual data points overlaid. Paired two-tailed Student’s t-test. d, Percentages of SPON2⁺ NK and SPON2⁻ NK cells with NK-immunity-related pathways enriched (Pct. exp); average expression (Ave. exp). e, IFN-γ receptor gene expressions at TC. We separated SPON2^high (n = 3 ROIs) and SPON2^low (n = 4 ROIs) subgroups by SPON2’s median expression of CD57^high ROIs (23.84). IFNGR1 (P = 0.016), IFNGR2 (P < 0.0001). Unpaired two-tailed Student’s t-test. f, Projections of enrichment scores based on SPON2, ZFP36, ZFP36L2, VIM, or HLA-DRB1, in combination with NK marker genes (see Methods). These enrichment scores (coded by color gradients) are projected onto the AS, IF, and TC compartments of tissues from REC or non-REC patients. From left to right panels, the enrichment scores denote the abundances of SPON2⁺ NK, ZFP36⁺ NK, ZFP36L2⁺ NK, VIM⁺ NK, and HLA-DRB1⁺ NK cells, respectively. g, Gene ontology (GO) enrichment analysis identifies biological pathway differences between SPON2⁺NK^high and SPON2⁺NK^low spots at TC. The pathways showing most significant upregulations in SPON2⁺NK^high spots compared to SPON2⁺NK^low spots at TC are listed. h, Ridge plots comparing IFNGR2 expression in SPON2⁺NK^high and SPON2⁺NK^low at TC. The upper and lower panels show the distributions of IFNGR2 expression levels in SPON2⁺NK^high and SPON2⁺NK^low spots at TC, respectively. Color gradient depicts the probability density. i, Gene set enrichment analysis for positive regulation of leukocyte migration and leukocyte mediated immunity at TC, based on differentially expressed genes between SPON2⁺NK^high and SPON2⁺NK^low spots. NES: normalized enrichment score; statistical significance through a one-tailed permutation test (see Methods). j, Gene set variation analysis compares immune processes between SPON2^highCD57^high and SPON2^lowCD57^high ROIs at TC. The listed immune processes highlight distinctions between SPON2^highCD57^high (n = 3 ROIs) and SPON2^lowCD57^high (n = 4 ROIs) subgroups, categorized by the median expression level of SPON2 at CD57^high TC (23.84). The columns denote the ROI samples. Color gradient depicts the enrichment level. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Source data