**Anna Sophie Decker 1, Ekaterina Pylaeva 1, Alexandra Brenzel 2, Ilona Spyra 1, Freya Droege 1, Timon Hussain 1, Stephan Lang <sup>1</sup> and Jadwiga Jablonska 1,\***


Received: 1 January 1970; Accepted: 19 August 2019; Published: 21 August 2019

**Abstract:** Neutrophil extracellular traps (NETs) represent web-like structures consisting of externalized DNA decorated with granule proteins that are responsible for trapping and killing bacteria. However, undesirable effects of NET formation during carcinogenesis, such as metastasis support, have been described. In the present study, we evaluated the correlation between NETosis and disease progression in head and neck cancer (HNC) patients in order to establish a valid biomarker for an early detection and monitoring of HNC progression. Moreover, factors influencing NET release in HNC patients were revealed. We showed a significantly elevated vital NETosis in neutrophils isolated from early T1–T2 and N0–N2 stage patients, as compared to healthy controls. Additionally, in our experimental setting, we confirmed the involvement of tumor cells in the stimulation of NET formation. Interestingly, in advanced cancer stages (T3–4, N3) NETosis was reduced. This also correlated with the levels of granulocyte colony-stimulating factor (G-CSF) in plasma and tumor tissue. Altogether, we suggest that the elevated NETosis in blood can be used as a biomarker to detect early HNC and to predict patients at risk to develop tumor metastasis. Therapeutic disruption of NET formation may offer new roads for successful treatment of HNC patients in order to prevent metastasis.

**Keywords:** head-and-neck cancer; metastasis; neutrophils; NETs; NETosis; innate immunity; G-CSF

#### **1. Introduction**

Head and neck cancer (HNC) is one of the most common tumor entities worldwide with an incidence of around 680,000 new cases per year [1]. The majority of HNC is histologically diagnosed as squamous cell carcinomas. At the time of diagnosis most HNC are locally advanced, which, despite multimodal treatment, often leads to disease progression or recurrence [2]. Recurrent HNC is often no longer accessible to curative therapy, turning HNC into the ninth deadliest cancer disease [1]. Although some risk factors associated with bad prognosis are well known, it still remains unclear which patients will suffer from recurrent HNC. Thus, this field is lacking a noninvasive diagnostic tool for early HNC diagnosis and for monitoring recurrent HNC progression.

Neutrophils play an important role in innate immunity, as they are the first line of defense during acute infection. Neutrophils show various strategies to protect the body from intruding microbes, fungi, and other pathogens [3]. Besides the well-known mechanisms like phagocytosis, degranulation, or production of reactive oxygen species (ROS), another antimicrobial mechanism of neutrophils has been described—neutrophil extracellular trap (NET) formation. NETs are web-like structures composed of externalized DNA decorated with histone and granule proteins. They can be released upon different stimuli, such as bacteria, cytokines, or the protein kinase C activator phorbole-12-myristat-13-acetat (PMA), resulting in pathogen killing [4]. Intensive investigations on NETs over the last decade revealed a potential involvement of the NETs in cancer progression, as it was shown that tumor cells

themselves can activate neutrophils and stimulate NET formation via production of different factors like granulocyte colony-stimulating factor (G-CSF) [5]. Moreover, it was observed that released NETs, produced upon stimulation via tumor cells, are able to capture and surround distant metastatic cells and circulating tumor cells and, so, promote metastatic processes [6]. NETs were also shown to support invasion and migration of tumor cells in vitro. Degradation of NETs by DNase treatment prevented metastasis in a murine tumor model [7].

While localized tumors can be easily removed, metastatic disease remains the leading cause of death among cancer patients. Therefore, since NET formation is supposed to facilitate metastasis, we wanted to evaluate if there is a correlation between disease progression in HNC patients and NET formation by blood neutrophils. Moreover, we wanted to reveal factors that are involved in this phenomenon. This should help to reveal useful prognostic biomarkers to identify patients prone to HNC metastasis.
