Abstract 5942: Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma

The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. Pro-inflammatory cytokines regulated by NF-κB promote extravasation of neutrophils into tissue; neutrophils are polarized to anti-tumor (N1) or pro-tumor (N2) phenotype through the action of various pathways, including TGF-β. The mechanism and role of neutrophil recruitment in HNSCC were explored this study. Neutrophils isolated from adult donors were co-cultured for 5 hours with conditioned media (CM) from HNSCC cells SCC-9 and Cal-27, and stained with TNF-α, ICAM-1, CCL2, CCL5 for analysis of neutrophil phenotype by flow cytometry. TGF-β blocking antibody was used to explore the role of TGF-β in polarization of neutrophils. Canonical and non-canonical TGF-β signaling pathway activation was also investigated in these neutrophils through western blots. Using HNSCC patient samples, qPCR was performed for expression of genes involved in apoptosis, epithelial-to-mesenchymal transition, metastasis, and neutrophil function. Following co-culture with 24-hour HNSCC CM, neutrophils begin to exhibit cell surface markers indicative of N2 phenotype, which is impacted by TGF-β blocking antibody. Neutrophils co-cultured with HNSCC CM exhibited altered expression of TGF-β mediators Smad2/3, p38 and Akt, which was abrogated with the addition of TGF-β blocking antibody indicating specificity of a TGF-β induced signaling. Gene expression analysis exhibited significantly higher expression of anti-apoptotic BCL2 and neutrophil function marker alpha defensin 1 in HNSCC patient samples with greater neutrophil infiltration, while caspase 3 was expressed at significantly higher levels in HNSCC patient samples with low neutrophil infiltration. HNSCC cells recruit neutrophils; evidence suggests that TGF-β pathway, which typically promotes N2 phenotype in neutrophils, is involved via canonical and non-canonical mediators including p38 and Akt. Further, HNSCC patient samples have varying levels of neutrophil infiltration and differentially express genes involved in apoptosis, EMT, metastasis and invasion, inflammation. Citation Format: Kulsoom Ghias, Hina Shakeel, Alisalman Sheikh, Syed Hani Abidi, Kiran I. Masood, Saira Fatima, Fareena Bilwani. Mechanism and role of neutrophil recruitment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5942.