**2. Materials and Methods**

#### *2.1. Patients*

Patients with HNC (group 1, *n* = 36, blood samples for neutrophil isolation; group 2, *n* = 17, whole blood samples for SYTOX staining; group 3, *n* = 20, tumor samples) and healthy volunteers (*n* = 10) were included in the study after written local ethics committee approval; no previous chemotherapy or radiotherapy was performed. Acute inflammatory events (infectious diseases or acute phase of autoimmune disorders) 6 months prior to the enrollment were the exclusion criteria for this study. Clinico-pathological characteristics of HNC patients enrolled in this study are listed in Table 1.


**Table 1.** Clinico-pathological characteristics of patients enrolled in this study. HNC—head and neck cancer.

\* Oral cavity, glands, nasopharynx, hypopharynx.

#### *2.2. Isolation of Blood Neutrophils*

Peripheral blood was drawn into 3.8% sodium citrate anticoagulant monovettes and separated by density gradient centrifugation (Biocoll density 1.077 g/mL, Biochrome). The mononuclear cell fraction was discarded, and neutrophils were isolated by sedimentation over 1% polyvinyl alcohol, followed by hypotonic (0.2% NaCl) lysis of erythrocytes. In view of the emerging diversity of circulating neutrophil subtypes in humans, it should be noted that high-density neutrophils were investigated in this study.

The purity of the isolated neutrophils (>95%) was estimated with flow cytometry after staining with anti-CD66b (Beckman Coulter, Krefeld, Germany). Viability Dye eFluor™ 780 (eBioscience, Affymetrix, Santa Clara, CA, USA) or 4 ,6-Diamidino-2-Phenylindole, Dilactate (DAPI) (BioLegend, San Diego, CA, USA) were used to determine viable cells. (Supplementary Figure S1A). Data were collected and analyzed with the BD FACS Canto system and BD FACS Diva 6.0 software (BD Biosciences, BD, Franklin Lakes, NJ, USA).

#### *2.3. Bacteria*

To stimulate NET release, *Pseudomonas aeruginosa* strain PA14 (wild-type serogroup O10 strain, cytotoxic ExoU+) was used. Bacteria were cultured in Luria–Bertani (LB) broth for 3 h to reach the early exponential phase, washed twice in phosphate buffered saline (PBS), and the optical density of a 100 μL suspension was measured in 96-well flat-bottom cell culture plates (Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany) at 600 nm using a microplate reader Synergy 2 (BioTek Instruments, Inc., Winooski, VT, USA). OD 0.4 corresponds to a bacterial density of 5 <sup>×</sup> <sup>10</sup>9/mL, as determined by serial dilutions and colony-forming unit assays. Bacteria concentration was adjusted to the desired values and verified by further plating on 2% LB agar plates.

### *2.4. Head and Neck Cancer (HNC) Tumor*

Tumor tissue was digested using dispase 0.2 μg/mL, collagenase A 0.2 μg/mL, and DNase I 100 μg/mL (all Sigma-Aldrich/Merck, Darmstadt, Germany) solution in DMEM (Gibco, Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FCS) and 1% penicillin–streptomycin to exclude the influence of live bacteria on NET formation by neutrophils. Cells were meshed through 50 μm filters (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany), and the concentration was measured with a CASY cell counter (Innovatis, Roche Innovatis AG, Bielefeld, Germany).

The percentage of tumor-associated neutrophils from single live cells in tumor tissue was estimated in a single-cell suspension after staining with anti-CD66b (Beckman Coulter) and Viability Dye eFluor™ 780 (eBioscience, Affymetrix).

For tumor supernatant isolation, tumor weight was measured, the tissue was cut into 0.5–1 mm pieces, and the amount of medium (DMEM (Gibco, Life Technologies/Thermo Fisher Scientific) containing 10% FCS and 1% penicillin–streptomycin) was added accordingly by weight (0.6 mL per 0.02 g). The samples were incubated for 4 h at 37 ◦C, 5% CO2, and sterile medium was used as a negative control.
