Single-cell transcriptomics reveals that tumor-infiltrating natural killer cells are activated by localized ablative immunotherapy and share anti-tumor signatures induced by immune checkpoint inhibitors.

Rationale: Natural killer (NK) cells provide protective anti-cancer immunity. However, the cancer therapy induced activation gene signatures and pathways in NK cells remain unclear. Methods: We applied a novel localized ablative immunotherapy (LAIT) by synergizing photothermal therapy (PTT) with intra-tumor delivering of the immunostimulant N-dihydrogalactochitosan (GC), to treat breast cancer using a mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mouse model. We performed single-cell RNA sequencing (scRNAseq) analysis to unveil the cellular heterogeneity and compare the transcriptional alterations induced by PTT, GC, and LAIT in NK cells within the tumor microenvironment (TME). Results: ScRNAseq showed that NK subtypes, including cycling, activated, interferon-stimulated, and cytotoxic NK cells. Trajectory analysis revealed a route toward activation and cytotoxicity following pseudotime progression. Both GC and LAIT elevated gene expression associated with NK cell activation, cytolytic effectors, activating receptors, IFN pathway components, and cytokines/chemokines in NK subtypes. Single-cell transcriptomics analysis using immune checkpoint inhibitor (ICI)-treated animal and human samples revealed that ICI-induced NK activation and cytotoxicity across several cancer types. Furthermore, ICI-induced NK gene signatures were also induced by LAIT treatment. We also discovered that several types of cancer patients had significantly longer overall survival when they had higher expression of genes in NK cells that were also specifically upregulated by LAIT. Conclusion: Our findings show for the first time that LAIT activates cytotoxicity in NK cells and the upregulated genes positively correlate with beneficial clinical outcomes for cancer patients. More importantly, our results further establish the correlation between the effects of LAIT and ICI on NK cells, hence expanding our understanding of mechanism of LAIT in remodeling TME and shedding light on the potentials of NK cell activation and anti-tumor cytotoxic functions in clinical applications.