Background Head and neck squamous cell carcinomas (HNSCC) are the sixth most common cancer worldwide and result in 400,000 deaths annually.1 Interleukin-9 (IL-9) is a pleiotropic cytokine largely produced by CD4+ Th9 cells.2 IL-9 is known to function in allergic inflammatory processes and is involved in positive and negative regulation of the immune response. The role of IL-9 in cancer differs depending on the cancer type and the tumor microenvironment, harboring both tumorigenic and anti-tumorigenic effects.3–8 However, the role of IL-9 is poorly understood in HNSCC. Here, we begin to elucidate possible functions of IL-9 in HNSCC.
Methods IL-9 plasma and PBMC levels were determined by ELISA and cell proliferation was measured using a metabolic absorbance assay. Immune cell infiltration was determined by co-culturing Cal27 spheroids with HNSCC PBMCs, dissociating the spheroids, and analyzing the percentage of live infiltrating PBMCs by flow cytometry.
Results IL-9 plasma levels were found to be significantly increased in HNSCC patients as compared to healthy counterparts, and higher pre-treatment plasma levels of IL-9 in HNSCC patients were associated with tumor recurrence. IL-9 did not affect tumor cell proliferation in vitro. However, higher doses of IL-9 significantly decreased immune cell infiltration into Cal27 spheroids. When natural killer (NK) cells were depleted from peripheral blood mononuclear cells (PBMCs), there was a significant increase in IL-9 in both HNSCC PBMCs and healthy counterpart PBMCs.
Conclusions IL-9 did not directly enhance or inhibit HNSCC cell proliferation. Importantly, IL-9 significantly decreased immune cell infiltration into HNSCC spheroids, suggesting that IL-9 inhibits immune cell infiltration into the tumor in vivo. Because IL-9 levels were increased when NK cells were depleted from PBMCs, it is likely that NK cells are inhibiting the production or the release of IL-9. Future studies will focus on the effects of IL-9 on immune cell cytotoxicity, immune infiltration in vivo, and elucidating the inhibitory effects of NK cells on IL-9 production/release.
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Ethics Approval Human samples were collected as part of University of Cincinnati IRB approved protocol #2014–4755 after informed consent.
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