Role of interleukin-17f in oral tongue squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a group of common and aggressive malignant tumours that arise in the oral cavity, oropharynx, hypopharynx and larynx. The majority of HNSCC tumours are encountered in the oral cavity, where the tongue is the most frequently affected site intraorally (oral tongue SCC; OTSCC). The multimodality treatment includes surgery and radiotherapy with or without chemotherapy. In addition, some immunotherapeutic agents have been recently approved to treat HNSCC patients. However, patients with HNSCC in general, and those with OTSCC in particular, have a relatively unfavourable prognosis. Unfortunately, five-year overall survival rates in the Nordic countries including Finland remain between 50-60%. Therefore, there is an urgent need to identify new prognostic and therapeutic targets to improve the survival outcomes of OTSCC patients. Interleukin‐17F (IL‐17F) is the latest identified member of the IL‐17 cytokine family. IL‐17F was recently shown to induce antitumorigenic effects in several cancer types through different mechanisms. The serum level of IL-17F was lower in OSCC patients compared with leukoplakia patients and healthy individuals. Furthermore, IL‐17F inhibited tumour angiogenesis and the level of IL-17F was significantly lower in tumour tissues such as in colon and hepatocellular carcinoma (HCC) compared with the healthy control samples. Overall, these reports suggest an inverse relationship between IL‐17F levels and tumorigenesis. The aims of the present doctoral thesis were set as follows: 1) to investigate the expression levels and cellular sources of IL‐17F in OTSCC tissues and its potential association with patients' prognosis and mortality outcomes; 2) to assess the effects of IL-17F on several tumorigenic features including cell proliferation, migration, invasion and angiogenesis; 3) to test the possible effect of IL-17F on the formation of tumour-derived de novo vessel-like structures (vasculogenic mimicry; VM); 4) based on the antiangiogenic effect of IL-17F, we also aimed to systematically review the prognostic value of blood microvessel density (MVD) and lymphatic vessel density (LVD) in patients with tongue SCC (TSCC). The studies included clinical samples obtained from OTSCC patients who were treated surgically at Oulu and Tampere University Hospitals during the period from 1990‐2016. The study was approved by the relevant Ethics Committees. In addition, different OTSCC cell lines, human umbilical vein endothelial cells (HUVEC), cancer-associated fibroblasts (CAF), and human oral keratinocytes (HOKs) were used for the in vitro assays. Gene analysis assays were performed using RT-PCR and droplet-digital PCR. Immunostaining and blinded-scoring were used to map IL-17F expression in patient samples. The IncuCyte system, tumour spheroids, FACS analysis, and tube-formation assays were also used. Survival curves were constructed according to the Kaplan-Meier method. We also tested different patterns of VM structures in patient samples and in an orthotopic mouse model of OTSCC. The systematic review search was conducted in the following databases: Ovid Medline, Scopus, and Cochrane libraries. Briefly, we first showed that mast cells are the main source of IL-17F in OTSCC. Using multivariate analysis, the extracellular mast cell-derived IL-17F at the tumour invasion front was associated with better disease-specific survival in all-stages and early-stages OTSCC patients. We also showed that OTSCC cells had lower levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with normal HOKs. Importantly, IL-17F inhibited tumour cell proliferation, random migration, and CAF-mediated tumour cell invasion. The tube formation by HUVEC was inhibited by IL-17F in a dose-dependent manner. Additionally, we showed that OTSCC cells contained CD31 at both mRNA and protein levels, and formed tube-like VM on Matrigel similar to those formed by HUVEC. These VM structures were also identified in a mouse orthotopic model of human OTSCC. Furthermore, IL-17F supressed the formation of VM in vitro, which mimics the effect produced by the anti-angiogenic drug Sorafenib. Finally, our systematic review results revealed that higher expression of MVD/LVD markers were associated with worse survival outcomes in TSCC patients in most of the relevant studies. Taken together, our findings support an anticancerous role of IL-17F in OTSCC. Based on levels of extracellular mast cell-derived IL-17F at the tumour invasive front, patients with OTSCC were categorized into high- and low-risk groups. The in vitro assays revealed inhibitory effects of IL-17F on tumour cell proliferation, migration, invasion and VM formation, and thus further preclinical studies are warranted. We also concluded that more clinical studies with larger patient cohorts are needed before the MVD/LVD markers can be recommended for clinical prognostication. Development of IL-17F-based drugs, or targeting one of its regulatory pathways, may have prognostic implications as well as serve as a promising therapeutic approach in patients with OTSCC .