Trojan horse natural killer cells enhance targeted drug delivery and boost memory T cell-mediated immune responses in triple-negative breast cancer.

Natural killer (NK) cell-based cancer therapy is a key innovation in immunotherapy. However, current research on drug delivery systems (DDSs) mediated by NK "dead cells" remains limited. Accordingly, we developed an innovative method to obtain dead NK cells as drug carriers using rapid vitrification freezing and slow-programmed thawing. Freeze-thawed NK cells (FTKs) showed enhanced tumor-targeting ability by upregulating the expression of C-X-C motif chemokine receptor 3 and integrin beta 2. Therefore, FTKs function as Trojan horse-like carriers, enabling efficient delivery of toxic chemotherapeutic agents. In addition, FTKs were also found to stimulate the maturation of dendritic cells (DCs) via the release of danger-associated molecular patterns. In conjunction with chemotherapy, this strategy can synergistically leverage the immunomodulatory properties of chemotherapeutic drugs to increase immunogenic cell death, mitigate immunosuppression within the tumor microenvironment, and slow the progression of triple-negative breast cancer (TNBC). Using a metastatic TNBC mouse model, this strategy enhanced the immunological effect of a programmed cell death protein 1 inhibitor by re-invigorating cytotoxic T lymphocytes and promoting the reacquisition of memory T-cell responses. Hence, we propose an NK "dead cell"-based enhanced target delivery system that can be rapidly manufactured for clinical use, thus providing insights into innovative immunotherapy drug delivery strategies. STATEMENT OF SIGNIFICANCE: Triple-negative breast cancer is a highly aggressive subtype of breast cancer, for which chemotherapy is the primary treatment modality. However, conventional chemotherapy is often limited by insufficient target specificity, leading to systemic toxicity. In this study, we developed a drug delivery platform based on natural killer (NK) cells. By processing cells via rapid vitrification freezing and slow-programmed thawing, we obtained freeze-thawed NK cells (FTKs) characterized by an intact membrane architecture and enhanced tumor-targeting capacity. In addition, FTKs were found to promote dendritic cell maturation and had a high drug-loading capacity. In conjunction with chemotherapy, this strategy enhanced the immunological effect of a programmed cell death protein 1 inhibitor by re-invigorating cytotoxic T lymphocytes and promoting the reacquisition of memory T-cell responses.