Extended Data Fig. 1: Integrative Analysis of Spatial Transcriptomics in HCC Tissue Compartments and NK Cell Protein Expression Related to HCC Recurrence.

a, Hematoxylin and Eosin (H&E), Glypican 3 (GPC3), CD34, glutamine synthetase (GS), and Reticulin staining to delineate HCC tissue compartments. Tissue compartments including invasive front (IF) and tumor center (TC) were outlined using black dashed lines. These high-resolution images of other cases have been uploaded to the Open Science Framework public platform (https://osf.io/db3uf/?view_only=d8431249989045bab050581751199298). Experienced hepatopathologists Y.Z. and J.Y. thoroughly re-reviewed our selected sections to confirm delineations of adjacent stroma (AS), IF, and TC across all cases utilizing established HCC markers. b, Visium transcriptome clustering patterns differ in various pathological regions within tissues. K-Means clustering results of 10x Visium samples independently (top panel) and their Uniform Manifold Approximation and Projection (UMAP, bottom panel). c, Integrated UMAP embedding of all spatial spots from donors. Integrating the UMAP clustering results of 18 Visium samples from 17 patients. Coloration distinguishes spatial compartments. d, Comparison of CD3⁻CD56⁺ NK cell proportions at IF between non-recurrent (non-REC; n = 31 patients) and current (REC; n = 30 patients) HCC patients. Unpaired two-tailed Student’s t-test, P = 0.035. This enabled delineation of CD3⁻CD56⁺ ordinary NK cells (shown here), CD3⁻CD16⁺CD56⁺ cytotoxic NK cells (shown in d), and CD3⁻CD57⁺ mature NK cells (shown in Fig. 1f) subsets at the single cell level. For non-REC, max (3.13608), min (0.05872), median (0.97038), mean (1.32004), Q1 (0.61261), Q3 (2.05738); For REC, max (2.80434), min (0.06898), median (0.82503), mean (0.89512), Q1 (0.48859), Q3 (1.29746). IQR = Q3-Q1, Upper Whiskers = Q3 + 1.5*IQR, Lower Whisker = Q1-1.5*IQR; the whiskers extending to the most extreme data points. e, Comparison of CD3⁻CD16⁺CD56⁺ NK cell proportions at IF between non-REC (n = 31 patients) and REC (n = 30 patients) patients. Unpaired two-tailed Student’s t-test, P = 0.003. For non-REC, max (0.603270), min (0.005111), median (0.150094), mean (0.183848), Q1 (0.053496), Q3 (0.253224); For REC, max (0.250404), min (0.003508), median (0.058713), mean (0.081717), Q1 (0.025158), Q3 (0.136488). IQR = Q3-Q1, Upper Whiskers = Q3 + 1.5*IQR, Lower Whisker = Q1-1.5*IQR; the whiskers extending to the most extreme data points. f, Disease-free survival (DFS) analysis of stratified HCC patients. CD3⁻CD56⁺NK^high (n_non-REC = 17 patients, n_REC = 14 patients) and CD3⁻CD56⁺NK^low (n_non-REC = 14 patients, n_REC = 16 patients) subgroups were separated using the median ratio of CD3⁻CD56⁺ NK abundance relative to DAPI⁺ cell abundance at IF (0.9245). Shaded areas correspond to 95% confidence intervals and central lines indicate medians. The survival curves of the two subgroups cannot be clearly distinguished, as the log-rank test determined this comparison to be not significant, with notation of ns. g, DFS analysis of stratified HCC patients. CD3⁻CD16⁺CD56⁺NK^high (n_non-REC = 19 patients, n_REC = 12 patients) and CD3⁻CD16⁺CD56⁺NK^low (n_non-REC = 12 patients, n_REC = 18 patients) subgroups were separated using the median ratio of CD3⁻CD16⁺CD56⁺ NK abundance relative to DAPI⁺ cell abundance at IF (0.0827). Shaded areas correspond to 95% confidence intervals and central lines indicate medians. The survival curves of the two subgroups cannot be distinguished with significance, as suggested by the log-rank test and marked as ns. Statistical significance: *P < 0.05, **P < 0.01, and ns—not significant.

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