Altered cytokine production can lead to immune dysfunction in patients with cancer. The present study investigated the expression of T helper (Th)17 cytokines in patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance in providing new therapeutic insights. The prevalence of Th17 cells and their receptors in patients with LSCC was studied using immunohistochemical analysis via tissue microarray technology. Flow cytometry was used to investigate the percentage of Th17 and Th1 cells in peripheral blood mononuclear cells. Furthermore, the proliferation of Th17 cells and Th17-associated cytokines, including interleukin (IL)17, IL23 and RAR-related orphan receptor γt, was analyzed by reverse transcription-quantitative polymerase chain reaction. The results revealed that the prevalence of Th17 cells in patients with LSCC was elevated in their primary tumors, as well as in peripheral blood, compared with that in healthy controls. It was further demonstrated that Th17 cells could be induced and expanded in the tumor microenvironment through cytokines produced by the tumor cells. In conclusion, Th17 cells have a substantial impact on the carcinogenesis of LSCCs, and could serve as a potential therapeutic target to modulate the anti-tumor response in these carcinomas.
Laryngeal carcinoma is the 11th most common form of cancer in humans worldwide (
Previous studies have confirmed that tumor immune inflammation is important in the tumor microenvironment (
The present study revealed that patients with LSCC have elevated levels of Th17 cells in their primary tumors and peripheral blood compared with those in healthy controls. In addition, the LSCC microenvironment was identified as a strong Th17-cell inducer.
A total of 70 tumors and 70 adjacent control tissues (pathologically confirmed normal mucosa) were obtained from patients with LSCC undergoing surgery. Peripheral blood was obtained from another 36 patients with LSCC and from 16 healthy individuals, who served as controls. The patients were registered for treatment at the Eye, Ear, Nose and Throat Hospital of Fudan University (Shanghai, China) from September 2013 to January 2015. For LSCC samples, patients with a known history of LSCC were enrolled, and malignancy was assessed by a pathological examination of the biopsies. The tumor staging of the patients was performed in accordance with the American Joint Committee on Cancer tumor-node metastasis (TNM) classification (
TMA technology is a method where a large number of tissue samples are placed on a microscopic glass slide, which facilitates the transition from basic research to clinical applications (
Two TMA blocks with 70 tumors and 70 adjacent tissues samples were used for immunostaining, followed by standard procedures for the avidin-biotin-peroxidase method (
The slices were evaluated by two pathologists without knowledge of the clinical outcome. The percentage of immunoreactive cells was graded on a scale of 0 to 4, as follows: No staining was scored as 0; 1–10% of stained cells was scored as 1; 11–50% of stained cells was scored as 2; 51–80% of stained cells was scored as 3; and 81–100% of stained cells was scored as 4. The staining intensities were graded from 0 to 3, as follows: 0 was defined as negative; 1 as weak; 2 as moderate; and 3 as strong. The raw data were converted into an immunohistochemical score (IHS) by multiplying the quantity and intensity scores. An IHS score of 9–12 was considered to represent strong immunoreactivity (+++); 5–8 was considered as moderate (++); 1–4 was considered as weak (+); and 0 was considered as negative immunoreactivity (−). On the final analysis, the cases that had an IHS <1 were considered as negative, and those with an IHS ≥1 were regarded as positive.
Peripheral blood mononuclear cells (PBMCs) were isolated from 5 ml of freshly obtained peripheral blood by centrifugation (800 × g, 20°C, 20 min) on a Ficoll Hypaque density gradient (Ficoll PM 400; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Prior to intracellular staining, the isolated PBMCs were stimulated for 5 h with 2 µl/ml Cell Stimulation Cocktail (#00-4970; eBioscience, Inc., San Diego, CA, USA), a cocktail of phorbol 12-myristate 13-acetate (PMA; eBioscience, Inc.) and ionomycin (eBioscience, Inc.) in the presence of Protein Transport Inhibitor Cocktail (#560751; BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were fixed and permeabilised using the BD Cytofix/Cytoperm™ Buffer (Fixation and Permeabilization Solution; #560751; BD Biosciences) according to manufacturer's protocol. Subsequently, the isolated PBMCs were intracellularly stained with the Human Th1/Th2/Th17 Phenotyping Cocktail (560751; BD Biosciences). Flow cytometry was performed on a BD FACSCalibur (BD Biosciences) and the data were evaluated using FlowJo software version 7.6.1 (TreeStar, Inc., Ashland, OR, USA). To determine the percentage of Th17 and Th1 cells, lymphocytes were gated by plotting forward vs. side scatter followed by gating on CD4+ T cells. The gated cells were then analyzed for IL-17A as phycoerythrin and interferon (IFN)-γ as fluorescein isothiocyanate expression.
Total RNA was extracted from patients' frozen tissues using TRIzol reagent (15596-018; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 1 µg total RNA in a 20-µl reaction system using PrimeScript RT Reagent kit (Perfect Real Time; DRR063A; Takara, Bio, Inc., Otsu, Japan). The cDNA was then diluted with sterile water and stored at −20°C. The RT procedure using the PrimeScript RT Reagent kit was performed according to the manufacturer's protocol.
RT-qPCR for IL17, IL23 and RAR-related orphan receptor (ROR) γt was performed on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.), and the data were analyzed using the Applied Biosciences Real-Tiem PCR system 7500 software version 2.0.6 (Applied Biosystems; Thermo Fisher Scientific, Inc.). In brief, 2 µl cDNA was added to a 20-µl reaction mixture containing 10 µl 2X SYBR Premix Ex Taq, 0.4 µl forward primer (10 µM), 0.4 µl reverse primer (10 µM), 0.4 µl ROX reference dye and 6.8 µl sterile water (all #DRR063A; Takara, Bio, Inc., Otsu, Japan). All primers were designed using the Primer Premier version 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), with their specificity confirmed by the Basic Local Alignment Search Tool on the National Center for Biotechnology Information website (
Relative gene expression was calculated using the comparative quantification cycle (Cq) method (
The data were reported as the mean ± standard deviation or the mean ± standard error. IL-17 and IL-17R expression in tumors and adjacent tissues was evaluated by χ2 test. The percentage of Th17 and Th17/Th1 cells in the peripheral blood of patients with cancer compared with that of healthy controls was assessed using t-tests. Cytokines IL17, IL23 and RORγt in LSCC tissues compared with that of non-cancerous tissues were assessed using one-way analysis of variance. All statistical calculations were performed using SPSS version 19 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistical significant difference.
To study the expression of IL17 and IL17R
Overall, positive staining for IL17 was noted in 57 (81.43%) of 70 tumors with the following scores: 0, 13 (18.57%) of 70; 1–4, 16 (22.86%) of 70; 5–8, 27 (38.57%) of 76; and 9–12, 14 (20.00%) of 70 samples. Among the controls, positive staining for IL17 was noted in 26 (37.14%) of 70 samples. IL17R positive staining was observed in 64 (91.43%) of 70 cases. These included 0, 6 (8.57%) of 70; 1–4, 22 (31.43%) of 70; 5–8, 28 (40.00%) of 70; and 9–12, 14 (20.00%) of 70 cases. Among the controls, positive staining for IL17R was noted in 49 (70.00%) of 70 samples (
The prevalence of Th17 cells in the peripheral blood of 36 patients with LSCC was compared with that of 16 healthy controls to address whether there was an increased prevalence of Th17 cells in patients with LSCC. PBMCs were isolated and stimulated with PMA and ionomycin for 5 h in the presence of Protein Transport Inhibitor, and Th17 and Th1 cells were next quantified by flow cytometry. Th17 cells were identified as a CD4+IL17+ cell population. Th1 cells were identified as a CD4+IFN-γ+ cell population. As shown in
Th17/Th1 cells, which simultaneously produce the Th17 cytokine IL17 and the Th1 cytokine IFN-γ (
RT-qPCR revealed that the Th17-associated intracellular cytokines and transcription factors IL17, IL23 and RORγt in LSCC tissues were upregulated compared with their levels in matched adjacent non-cancerous tissues (
Tumors grow in a complex and active microenvironment. Within or nearby the tumor nests, lymphocytes as well as endothelial, stromal and innate cells are present, which interact with each other to form the tumor microenvironment (
LSCCs produce various immunosuppressive and tumor-promoting cytokines, leading to an impaired anti-tumor response (
It was previously shown that Th17 cells are involved in tissue inflammation by inducing the release of various cytokines, including IL6, IL21, IL23, IL1β and TGF-β, by neighboring tumor cells, tumor-derived fibroblasts and antigen-presenting cells (
Several studies demonstrated that human Th17 cells produce IL17A and exhibit RORγt expression (
The present study revealed that patients with LSCC have elevated levels of Th17 cells in their primary tumors and peripheral blood compared with those exhibited by healthy controls. The LSCC microenvironment was identified as a strong Th17-cell inducer. First, histopathological characterization was performed, and immunohistochemistry staining was used to detect IL17 and IL17R expression in patients with LSCC. Overexpression of both IL17 and IL17R was observed in tumors compared with the expression detected in adjacent tissues. Next, whether Th17 cells have any functional implications for LSCC development was evaluated. The frequencies of Th17 and Th1 cells in the peripheral blood of patients with LSCC were investigated, and a higher percentage of Th17 cells was detected in the peripheral blood of patients with LSCC compared with that in healthy controls. No association was observed between TNM staging and Th17 cell frequency, which suggests that Th17 cells were consistently elevated in patients with LSCC, independently of tumor stage. This may represent a link between inflammation and cancer; however, the exact mechanisms by which elevated levels of pro-inflammatory Th17 cells and the resultant secretion of cytokines contribute to inflammatory processes in cancer remain to be elucidated.
The present study demonstrated that Th17 cells can be induced by PBMCs of patients with LSCC. Upon incubation of isolated naïve CD4+ T cells in LSCC PBMCs
RT-qPCR demonstrated that the levels of Th17-associated intracellular cytokines and transcription factors (including IL17, IL23 and RORγt) of LSCC tissue were upregulated. Since it was shown that IL23 and RORγt lead to Th17 expansion (
Altogether, the present study demonstrated that Th17 cells are highly present in LSCC. However, this appears to act as a double-edged sword: On one hand, Th17 cells accelerate metastasis and appear to be, therefore, beneficial to tumors; on the other hand, Th17 cells appear to be beneficial to the host due to their proliferation-reducing activity. Therefore, it is important to study the function of Th17 cells in malignant diseases in depth, and to attempt to elucidate their mechanism of action and their modulation by the tumor microenvironment. The present results raise a further issue to investigate, namely whether Th17 cells express different molecules or secrete different cytokines in patients with LSCC compared with those in healthy individuals. Future studies should consider whether Th17 cells can recognize tumor cells and if they are at all able to impair tumor growth or metastasis
The present study was supported by the National Natural Science Foundation of China (grant nos. 30801283 and 30972691), the Shanghai Science and Technology Development Funds (grant nos. 09QA1401000, 10QA1405900 and 14411961900), the Training Program of the Excellent Young Talents of the Shanghai Municipal Health System (grant nos. XYQ2011055 and XYQ2011015) and the Shanghai Municipal Science and Technology Foundation (grant no. 11JC1410802).
Multitumor TMA. (A) Section containing 70 tumors and 70 adjacent tissues samples. The diameter of each tissue spot is 0.6 mm. (B) Schematic layout of the TMA section. TMA, tissue microarray.
Positive and negative expression of IL17/IL17R immunohistochemistry in tissue microarray sections. (A) Positive and (B) negative expression of IL17. (C) Positive and (D) negative expression of IL17R. Original magnification, ×20. IL, interleukin; R, receptor.
Levels of Th17 and Th1 cells in peripheral blood mononuclear cells from patients with LSCC and healthy controls. SCC, side scatter; FSC, forward scatter; Th, T helper; LSCC, laryngeal squamous cell carcinoma; CD, cluster of differentiation; IL, interleukin; IFN, interferon; H, height.
Prevalence of Th17 and Th17/Th1 cells in the peripheral blood of patients with LSCC and healthy controls. (A) Comparison of the prevalence of Th17 cells in the peripheral blood of healthy controls and patients with LSCC (*P<0.05). The data are expressed as the frequency of Th17 cells in the lymphocyte population. The box plots show the median (middle line), 25th and 75th percentiles (box, and the extreme values (whiskers). (B) Prevalence of Th17/Th1 cells in the peripheral blood of patients with LSCC compared with that in healthy controls with (*P<0.05). The box plots show the median (middle line), 25th and 75th percentiles (box), and the extreme values (whiskers). Th, T helper; LSCC, laryngeal squamous cell carcinoma.
Reverse transcription-quantitative polymerase chain reaction analysis of the Th17-associated intracellular cytokines and transcription factors IL17, IL23 and RORγt in tumors and controls. *P<0.05. IL, interleukin; ROR, RAR-related orphan receptor; Cq, quantification cycle.
Characteristic features of the patients in the current study.
Characteristics | Patients (fresh tissue) (n=70), n (%) | Patients (blood) (n=36), n (%) | Controls (blood) (n=16), n (%) |
---|---|---|---|
Age, years | |||
Mean (range) | 60.63 (38–84) | 59.10 (43–73) | 59.50 (52–72) |
Sex | |||
Male | 69 (98.57) | 36 (100.00) | 16 (100.00) |
Female | 1 (1.43) | 0 (0.00) | 0 (0.00) |
Site | |||
Supraglottic | 23 (32.86) | 6 (16.67) | |
Glottic | 40 (57.14) | 26 (72.22) | |
Infraglottic | 7 (10.00) | 4 (11.11) | |
cT stage | |||
T1+T2 | 17 (24.29) | 25 (69.44) | |
T3+T4 | 53 (75.71) | 11 (30.56) | |
pN stage | |||
N0 | 33 (47.14) | 31 (86.11) | |
N1+N2 | 37 (52.86) | 5 (13.89) | |
Clinical stage | |||
Early stage (I+II) | 6 (8.57) | 26 (72.22) | |
Late stage (III+IV) | 64 (91.43) | 10 (27.78) | |
Smoking history | |||
Smokers | 62 (88.57) | 25 (69.44) | |
Non-smokers | 8 (11.43) | 11 (30.56) | |
Alcohol consumption | |||
Drinkers | 34 (48.57) | 17 (47.22) | |
Non-drinkers | 36 (51.43) | 19 (52.78) |
Positive results of IL17/IL17R immunohistochemistry in tumors and controls.
Molecule | Tumors, n (%) | Controls, n (%) | P-value |
---|---|---|---|
IL17 | 57 (81.43) | 26 (37.14) | <0.05 |
IL17R | 64 (91.43) | 49 (70.00) | <0.05 |
IL, interleukin; R, receptor.