A total of 40 patients with TSCC who were admitted to The Department of Oral and Maxillofacial Surgery, Second Affiliated Hospital of Jinzhou Medical University (Liaoning, China) between January 2017 and June 2018 were used in the present study. The cohort consisted of 24 males and 16 females, aged 40–76 years old, with a median age of 54 years. The inclusion criteria were that all patients were diagnosed with TSCC by pathology and no radiation therapy or chemotherapy was performed prior to biopsy. Patients with one or more of the following conditions were excluded: i) Infectious disease; ii) acute cardiovascular and cerebrovascular diseases; iii) rheumatic disease; iv) diabetes; and v) other tumors. The present study was approved by The Ethical Committee of Jinzhou Medical University (Liaoning, China). Informed consent was obtained from all patients included in the study. In addition, 16 healthy individuals with no statistical difference in age and sex were used as the control group. All the participants were informed and blood samples of patients were collected prior to clinical treatment.

A total of 3 ml venous blood of each participant was collected and heparin sodium (10 mg/ml, Sigma-Aldrich; Merck KGaA) was used as an anticoagulant. Half of each blood sample was centrifuged (200 × g; 10 min; room temperature) to prepare serum samples with the remainder used to prepare peripheral blood mononuclear cells (PBMCs). All blood samples were processed within 6 h of collection.

TSCC tissues were fixed (>24 h at room temperature) in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded samples were cut into 4 µm sections and resected specimens were dewaxed in xylene, rehydrated, washed in distilled water and then stained with hematoxylin and eosin at room temperature for 5 min. Pathological alterations of myocardial tissue were observed under a light microscope (magnification, ×200).

PBMCs were isolated from the blood samples of patients with TSCC and healthy controls using lysing buffer (BD Biosciences) according to the manufacturer's protocol. Briefly, 3 ml 1× lysing solution was added to 3 ml venous blood, samples were incubated on ice for 5 min, centrifuged at 200 × g for 5 min at room temperature and then, the supernatant was carefully aspirated. The PBMCs were resuspended in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences) and counted with the density of cells adjusted to 1×10⁶ cells/ml for cell culture or flow cytometry analysis.

The PBMCs were resuspended in PBS. For surface staining, fluorescein isothiocyanate-conjugated anti CD4 antibody (cat. no. 555346; BD Biosciences) and allophycocyanin-conjugated anti CD25 antibodies (cat. no. 555434; BD Biosciences) were used. The cells were incubated at room temperature for 20 min protected from light, washed with 1X PBS twice then fixed and permeabilized for 40 min at room temperature using a fixation/permeabilization kit (cat. no. 555028, BD Biosciences) according to the manufacturer's protocol. After fixation and permeabilization, a phycoerythrin (PE)-conjugated anti-Foxp3 antibody (cat. no. 72-5774-40; eBioscience; Thermo Fisher Scientific, Inc.) was added. The cells were incubated at room temperature for 45 min protected from light then washed twice. After discarding the supernatant, cells were resuspended in 300 µl PBS for analysis using a flow cytometer. The results were analyzed using FlowJo version 10.1 (Tree Star, Inc.).

PBMCs were resuspended to a density of 2×10⁶ cells/ml, supplemented with 10% heat-inactivated fetal bovine serum (HyClone; GE Healthcare Life Sciences) and treated with phorbol-12-myristate-13-acetate (PMA; 10 ng/ml; Sigma-Aldrich; Merck KGaA), ionomycin (0.5 µg/ml; Sigma-Aldrich; Merck KGaA), and Brefedin A (1 µl/ml; BD Biosciences) in a 37°C, 5% CO₂ incubator for 5 h. The cultured cells were harvested and stained for surface markers and intracellular IL-17a.

Surface staining was described as above. The cells were washed once with PBS then fixed and permeabilized for 40 min at room temperature using a fixation/permeabilization kit (cat. no. 555028; BD Biosciences) following the manufacturer's protocols. Following fixation and permeabilization, anti-IL-17a PE (cat. no. 560436; BD Biosciences) was added. The cells were incubated at room temperature for 45 min protected from light then washed twice. After discarding the supernatant, cells were resuspended in 300 µl PBS for flow cytometry. Data analysis was performed using FlowJo software (version 10.6.0; FlowJo LLC).

PBMCs were collected and total RNA was extracted with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was reverse transcribed from total RNA using a RT kit (cat. no. K1622; Thermo Fisher Scientific, Inc.), according to the manufacturers' protocol. qPCR was performed using SYBR Select Master mix (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The primer sequences were as follows: Foxp3 forward, 5′-GCAGCTCTCAACGGTGGAT-3′ and reverse, 5′-GGGATTTGGGAAGGTGCAGA-3′; RAR-related orphan receptor-γ (RORγt) forward, 5′-GCCAAGGCTCAGTCATGAGA-3′ and reverse, 5′-CCTCACAGGTGATAACCCCG-3′; and GAPDH forward, 5′-TGTTGCCATCAATGACCCCTT-3′ and reverse, 5′-CTCCACGACGTACTCAGCG-3′. The PCR thermocycling conditions were 50°C for 2 min, 95°C for 2 min, 95°C for 15 sec, and 60°C for 1 min, for a total of 40 cycles. The 2^−ΔΔCq method was used to calculate the relative gene expression (12).

Serum samples were isolated from the blood of patients with TSCC and healthy controls via centrifugation at 450 × g for 10 min at room temperature. IL-10 and IL-17a levels were detected using the BD^™ CBA Human Th1/Th2/Th17 Cytokine kit (cat. no. 560484; BD Biosciences) according to the manufacturer's protocol. IL-10 and IL-17a levels were analyzed using FCAP Array v3.0 software (BD Biosciences).

All data are presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc.). The data was compared using unpaired 2-tailed Student's t-test. One-way analysis of variance followed by Kruskal-Wallis test was used to compare differences amongst multiple groups. P<0.05 was considered to indicate statistical significance. Experiments were performed in triplicate.

Clinical characteristics of patients with TSCC are presented in Table I. According to the Tumor-Node-Metastasis (TNM) classification for TSCC [Union for International Cancer Control, UICC 2010, 7th Edition (13)], the patients were divided into four stages: Stage I (n=4); stage II (n=12); stage III (n=17); and stage IV (n=7). H&E staining demonstrated that all patients had squamous cell carcinoma (Fig. 1).

To investigate the function of the immune system in patients with TSCC, CD4+ T cells were detected using flow cytometry. The results showed that the expression of CD4+T cells decreased in all TSCC patients. The capacity of patients to express CD4+T cells was relatively low (Fig. 2A). The percentage of Treg cells in the peripheral blood of patients with TSCC was significantly increased compared with the control group (control, 1.70±0.44%; stage I+II, 2.37±0.58%; stage III, 3.64±0.61%; and stage IV, 3.92±0.77%; Fig. 2B). The patients with advanced staged cancer (III or IV) exhibited significantly increased expression compared with patients with early stage cancer (I + II). The percentage of Th17 cells in the peripheral blood of patients with TSCC was significantly increased compared with the control. The percentage of Th17 cells increased significantly in with stage of cancer (Fig. 2C). These results indicated that T cells and Th17 cells affected the development of TSCC.

Tregs primarily exert immunoregulatory effects by producing IL-10 and Th17 cells primarily serve a role by secreting IL-17. IL-10 and IL-17a were detected in the peripheral blood of patients with TSCC using CBA. The results demonstrated that IL-10 (control 1.90±0.52%; stage I + II, 2.87±0.82%; stage III, 5.04±1.53%; and stage IV, 5.57±1.78%) and IL-17a (control 1.04±0.25%; stage I + II, 1.93±0.52%; stage III, 2.48±0.56%; and stage IV, 2.77±0.29%) in peripheral blood of patients with TSCC was significantly higher compared with the control (Fig. 3), Patients with advanced stage cancer (III or IV) exhibited significantly increased expression compared with patients with early stage cancer (I + II). These results suggested that, in line with the alterations in Treg and Th17 cells, the serum levels of IL-10 and IL-17 were increased gradually in patients with TSCC.

Foxp3 and RORγt are transcription factors in Treg and Th17 cells, respectively. The levels of Foxp3 and RORγt in PBMCs from patients with TSCC were detected using RT-qPCR. The results demonstrated that the levels of Foxp3 (control 1.07±0.13%; stage I + II, 2.17±0.57%; stage III, 3.14±0.31%; and stage IV, 3.42±0.33%) and RORγt (control 0.99±0.12%; stage I + II, 1.87±0.27%; stage III, 2.34±0.34%; and stage IV, 2.25±0.23%) in peripheral blood of patients with TSCC were significantly higher compared with the control (Fig. 4). Expression in patients with advanced stages of TSCC (III or IV) was significantly higher compared with patients with early stage TSCC (I + II). These results were consistent with the changes in Treg cells and Th17 cells. These results further demonstrated that Treg cells and Th17 cells affected the development of TSCC.