Integrated profiling of RUNX3 in intratumoral NK cells activity through bulk and single-cell transcriptomic analysis

Natural killer (NK) cells are core components of innate antitumor immunity, the dysfunction of NK cells in the tumor microenvironment is a major obstacle to the antitumor efficacy. Runt-related transcription factor 3 (RUNX3) acts as a critical tumor suppressor and regulates immune cell function, while its biological role in NK cells remains largely unexplored. Herein, we investigated the interaction between RUNX3 and NK cells in tumor microenvironment.

The Cancer Genome Atlas (TCGA) database was utilized to determine the genetic alteration of RUNX3 in pan-cancer. TIMER and GEPIA website were used to evaluate the correlation between RUNX3 and immune cell infiltration. Single-cell RNA sequencing (scRNA-seq) analysis was applied to characterize RUNX3 expression and pseudotime trajectory in NK cells. In vitro experiments were further performed to validate RUNX3's role in regulating NK cell functions.

RUNX3 was significantly downregulated in lung adenocarcinoma and hepatocellular carcinoma tissues. Clinical analyses have demonstrated that defective RUNX3 expression was correlated with adverse prognosis. Immune infiltration analyses revealed that RUNX3 was positively associated with immune cell infiltration, particularly NK cells and CD8⁺ T cells. scRNA-seq indicated RUNX3 enrichment in intratumoral NK cells, and differential genes of RUNX3 were enriched in the MAPK signaling pathway. Pseudotime trajectory analysis indicated RUNX3 participated in NK cell differentiation. Moreover, RUNX3 overexpression enhanced NK cell viability, chemotactic capacity, cytotoxicity against tumor cells, and secretion of pro-inflammatory cytokines and granzyme B, while upregulating NK cell activation receptors.

Our findings identify RUNX3 as a key regulator of NK cell-mediated antitumor immunity in LUAD and LIHC, providing a novel molecular target for enhancing innate immune surveillance and developing targeted immunotherapies for the aggressive malignancies.