Recruitment of Tumour-Associated Neutrophils to Head and Neck Squamous Cell Carcinoma

Objectives: Objectives: The presence of tumour-associated neutrophils (TAN) has been associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC). The aim of this study was to identify factors that are responsible for neutrophil recruitment into HNSCC.
Methods: Methods: TAN numbers within human HNSCC was evaluated by immunohistochemical staining for neutrophil myeloperoxidase (MPO). Factors secreted by FaDu HNSCC multi-cellular tumour spheroids (MCTS) were analysed by cytokine array and ELISA. Peripheral blood neutrophils from healthy volunteers were used to measure neutrophil migration to factors identified from the array. The recruitment of neutrophils to MCTS was assessed overtime by flow cytometry in the absence and presence of small molecule inhibitors. FaDu xenograft mouse models were used to confirm the effect of these inhibitors on neutrophil recruitment in vivo.
Results: Results: MPO staining confirmed the presence of marked numbers of TAN in HNSCC compared to normal oral epithelium. TAN were most abundant in the tumour invading front and necrotic areas. HNSCC MCTS resemble in vivo tumours, displaying areas of hypoxia, necrosis and cell proliferation. Neutrophils migrated in a dose-dependent manner to recombinant CXCL8, CXCL1 and MIF and these chemoattractants were found in abundance in the conditioned medium of FaDu MCTS. The recruitment of neutrophils to FaDu MCTS was significantly inhibited when neutrophils were pre-treated with antagonists for CXCR2 and CXCR4, the receptors for CXCL8, CXCL1 and MIF respectively. Moreover, use of the specific MIF inhibitor, ISO-1 caused a dramatic reduction in the number of neutrophils recruited into FaDu MCTS. In addition, in vivo, ISO-1 significantly reduced the number of TAN in xenograft FaDu tumours.
Conclusions: Conclusions: Collectively, these data suggest that CXCL8, CXCL1 and MIF in particular are important in the recruitment of TAN into HNSCC. Inhibition of these facors represent a potential novel anti-cancer target.