Anti-tumor effects on tumor-infiltrating natural killer cells by localized ablative immunotherapy and immune checkpoint inhibitors: An integrated and comparative study using scRNAseq analysis.

Localized ablative immunotherapy (LAIT), a combination of photothermal therapy (PTT) and the immunostimulant glycated chitosan (GC), has demonstrated therapeutic efficacy in cancer treatment. However, its impact on the tumor microenvironment (TME), particularly on tumor-infiltrating natural killer (TINK) cells, remains to be fully elucidated. Using single-cell RNA sequencing (scRNAseq), we analyzed the transcriptional and functional modulations of TINK cells by LAIT in a mouse breast cancer model. Additionally, we investigated immune checkpoint inhibitor (ICI)-induced changes in NK cells across multiple cancer types and evaluated the clinical relevance of these transcriptional changes using The Cancer Genome Atlas (TCGA) database. ScRNAseq revealed five NK cell subtypes, with LAIT increasing the proportion of interferon-enriched NK cells and enhancing NK cell differentiation and cytotoxicity. Functional analyses demonstrated that LAIT upregulated activation, cytotoxic, and interferon pathway genes while downregulating immune-suppressive genes, effects largely driven by GC. Comparative analysis showed significant transcriptional overlap between ICI and LAIT, highlighting shared pathways in NK cell-mediated cytotoxicity and chemokine signaling. Prognostic models constructed from ICI- and LAIT-induced gene signatures effectively stratified breast cancer patients by survival risk, with LAIT-induced genes showing the highest predictive performance. Furthermore, higher NK cell proportions and the expression of key prognostic genes, such as PSME2, IGKC, and KLRB1, were associated with improved overall survival. LAIT and ICIs enhance NK cell-mediated antitumor responses via distinct yet complementary mechanisms, emphasizing their potential for synergistic use. These findings provide novel insights into NK cell modulation within the TME and support the development of combinatorial immunotherapy strategies.