Extended Data Fig. 10: Detection of tumor-infiltrating leukocytes in HCC patients using mass cytometry and graphical summary of the anti-tumoral roles of SPON2⁺ NK cells.

a, Flow cytometry gating strategy of NK cells. Numbers in the drawn gates indicate the cell percentages. This gating strategy was used to select CD3⁻CD56⁺ NK cells. b, Classification of SPON2⁺ NK and other NK cells according to the expression level of SPON2. We show t-SNE projection of SPON2⁺ NK and other NK cells detected by mass cytometry. Upon categorizing the immune cell types based on SPON2 expression status at the protein level, 48.9% of SPON2⁺ cells corresponded to NK cells (also see Fig. 5m,n). This finding suggests that NK cells are the primary immune cell type expressing SPON2 in the HCC tumor microenvironment, further supporting the potential role of SPON2⁺ NK cells in HCC progression and their possible therapeutic relevance. c, t-SNE projection depicting IFN-γ expressions in HCC tissues. Detected by mass cytometry, IFN-γ expression is projected onto a cell type map that is the same as shown in the left panel of Fig. 5m. d, SPON2 expression in TC samples is positively correlated with patients’ overall survivals. The X-axis shows SPON2⁺ NK enrichment levels at TC, and the Y-axis denotes patients’ overall survivals in months. Spearman’s correlation coefficient ρ and P values are annotated (ρ = 0.59, P = 0.034), and a linear regression model is obtained and illustrated as a dashed red line (n = 13 samples from 13 patients, shown as blue points). Shaded areas correspond to 95% confidence intervals. e, The expression of SPON2 in NK cells is positively correlated with the expression of IFN-γ. The X-axis shows SPON2⁺ NK enrichment levels at IF and TC, and the Y-axis denotes IFN-γ+ NK enrichment levels at IF and TC. Two-tailed Spearman’s correlation coefficient ρ and P values are annotated, and linear regression models are obtained and illustrated as dashed red lines (n = 43 samples from 27 patients, shown as blue points). The correlation analysis suggested consistent trends between SPON2⁺ NK enrichment in HCC and IFN-γ production (ρ = 0.55, P = 1.6 × 10⁻⁴). Shaded areas correspond to 95% confidence intervals. f, Graphical summary of the anti-tumoral roles of SPON2⁺ NK cells. SPON2⁺ NK cell enrichment in the invasive front (IF) compartment of HCC tissue is associated with a lower risk of recurrence. These SPON2⁺ NK cells demonstrate functional activation with increased migration potential and cytotoxicity through elevated expression of IFN-γ and Perforin. Besides their direct cytotoxic activity, SPON2⁺ NK cells also interact with CD8⁺ T cells in anti-tumoral process. Therefore, HCC patients with a higher proportion of SPON2⁺ NK cells are expected to exhibit a lower risk of HCC recurrence.

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