Abstract
Tumor-associated macrophages (TAMs) and other PD-L1-expressing immune cells play a key role in head and neck squamous cell carcinoma (HNSCC). As PD-1 inhibitors have become standard therapy for recurrent/metastatic disease and are now used perioperatively in locally advanced resectable cases, understanding their impact on TAMs dynamics is critical. This study investigated the association between clinical features and TAMs prevalence in HNSCC, and their potential role in tumor progression and resistance to PD-1 blockade. Tumor samples from HNSCC patients were analyzed by simplex and multiplex immunohistochemistry, and fresh tumor slices were cultured ex vivo with PD-1 inhibitors. CD68 + and CD163 + macrophages and PD-L1 expression were quantified and correlated with clinical parameters. In a tissue microarray cohort of 96 patients, CD68 + and CD163 + macrophages were more abundant in oral and oropharyngeal tumors compared to laryngeal and hypopharyngeal carcinomas (p = 0.001 and p = 0.06, respectively). In 10% of cases, TAMs formed a barrier between tumor nests and immune infiltrates. Among nine patients with matched pre- and post-immunotherapy samples, TAMs density significantly increased post-treatment (p = 0.01 and p = 0.03), with CD163 + macrophages clustering at the tumor periphery in 78% of cases. Multiplex staining confirmed this spatial reorganization. Ex vivo exposure to PD-1 inhibitors reproduced the increase in TAMs. These results suggest that PD-1 blockade may foster M2-like macrophage accumulation, potentially contributing to immune evasion. This underscores the need for prospective studies and the development of macrophage-targeted strategies, particularly in the emerging context of perioperative immunotherapy in HNSCC.