Fig. 2: Single-cell expression profiles of tumor-infiltrating NK cells from aggressive sMIC⁺ tumors display distinct pro-inflammatory functional phenotypes with diminished cytotoxicity.

Tumors from sMICB^hi and sMICB^lo TRAMP/MIC mice were harvested, and single-cell suspension was obtained using enzymatic digestion. CD45⁺ tumor-infiltrating lymphocytes were isolated by cell sorting. Single-cell RNA-sequencing analysis of tumor-infiltrating lymphocytes was conducted using the 10× genomics platform. a Representative images of (left panel) sMICB^lo well-differentiated (WD) and (right panel) sMIC^hi poorly differentiated (PD) tumors from TRAMP/MICB mice. b Serum levels of sMICB in WD tumors (n = 6) and PD tumors (n = 4) from TRAMP/MICB mice. c UMAP plots illustrating different tumor-infiltrating immune cell populations identified by clustering algorithms in well-differentiated sMICB^lo and poorly differentiated sMICB^hi tumors. d Heatmap representing key gene ontology (GO) pathways affected in NK cells in sMICB^lo and sMICB^hi tumors identified using gene set enrichment analysis (GSEA). e UMAP plots illustrating identified NK cell cytotoxic and pro-inflammatory clusters in the sMICB^lo and sMICB^hi tumors. f Heatmaps representing the gene expression of cytotoxic and pro-inflammatory markers at the single NK cell level in the identified NK cell cytotoxic sub-cluster (top panel) and pro-inflammatory sub-cluster (bottom panel) in the sMICB^lo and sMICB^hi tumors. g Box plots representation of average cytotoxic marker gene expression level (top panel) and average pro-inflammatory marker gene expression level (bottom panel) in NK cells and their comparison between sMICB^lo and sMICB^hi tumors. h Functional analysis of tumor-infiltrating NK cells from sMICB^lo and sMICB^hi tumors in response to ex vivo PMA/Ionomycin stimulation assessed by flow cytometry. Representative flow cytometry dot plots and histograms demonstrating higher NK1.1⁺ cell population, higher IFNγ, Granzyme B, and TNFα-producing NK cells from sMICB^lo tumors compared to sMICB^hi tumors. **P < 0.01 (Student’s t test; two-tailed).