The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5‐year overall survival rate of around 50%, stagnant for decades. A tumour‐induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil‐to‐lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD‐L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC‐derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro‐tumour phenotype of NØ. UMSCC47‐TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD‐L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro‐tumour phenotype, dependent on ADO receptors (P1R), increasing CD170 high subpopulation, CD73 and PD‐L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD‐L1 expression. Co‐culture experiments with HNSCC cells demonstrated that TEX‐modulated NØ increase the CD73/PD‐L1 axis, through Cyclin D‐CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX‐mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies.