(A644) Transcriptional Profiling of Natural Killer Cells Reveals Dysfunction and Immune Suppression in Metastatic HNSCC

Introduction: Head and neck squamous cell carcinomas (HNSCC) are among the most common cancers worldwide, with patients diagnosed at advanced stages have a 5-year overall survival rate of 10-50%. Despite improvements in treatment, options for patients with recurrent or metastatic disease remain limited. Natural Killer (NK) cells play a pivotal role in tumor surveillance and control, but in HNSCC, they are especially susceptible to various tumor immune evasion strategies. The transcriptional and functional changes NK cells undergo in the immunosuppressive tumor microenvironment are not fully understood. To investigate this, we performed single-cell RNA sequencing (scRNA-seq) on NK cells isolated from regional metastatic and non-metastatic lymph nodes in HNSCC patients.

Methods: Metastatic and non-metastatic lymph nodes were collected from two patients undergoing HNSCC resection, processed into single-cell suspensions, and analyzed using scRNA-seq. After filtering and clustering based on gene expression patterns, we used module scores of differentially expressed genes to characterize distinct NK cell subsets.

Results: We identified six distinct NK cell subsets through unbiased clustering, visualized using a uniform manifold approximation and projection plot. The metastatic lymph nodes had significantly higher levels of functionally immature NK cells expressing suppressor genes such as KLRC1, TIGIT, and LAG3. In contrast, regional non-metastatic lymph nodes exhibited higher levels of functionally mature and memory NK cells expressing activating receptors like FCG3RA, KLRC2, and KLRK1. Finally, we defined a set of transcription factors upregulated in NK cells from metastatic lymph nodes.

Conclusions: Our studies reveal distinct transcriptional profiles of NK cells in metastatic versus non-metastatic lymph nodes in HNSCC, highlighting the role of functional immaturity and immune suppression in metastatic microenvironments. These findings could support the development of NK cell-based immunotherapies aimed at overcoming the immune evasion strategies utilized by HNSCC.