These authors have contributed equally to this work
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Lung cancer has been the leading cancer in the world, accounting for about 13% of all cancer cases. More seriously, its cancer mortality ranks first, leading to more than 1.4 million deaths per year worldwide. Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer, covering 80–85% of lung cancer cases. NSCLC patients are poorly responsive to conventional therapeutic strategies like surgery, chemotherapy and radiotherapy, with the 5-year survival of lower than 15% [
IL-17 is a class of T cell-derived proinflammatory cytokines, which is mainly produced by CD4+ T cells (Th17), CD8+ T cells (Tc17) and γδT (γδT17) cells. The IL-17 family consists of six members, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F. IL-17A is often referred to IL-17 due to the highest homology. It is widely involved in infectious diseases, autoimmune diseases and tumors via upregulating intercellular adhesion molecule-1 (ICAM-1), mediating the infiltration of inflammatory cells and T lymphocytes, and producing antibodies with the synergistic assistance of cytokines. Previous studies have shown that IL-17A can promote tumor immunosuppression and induce immune escape in colorectal cancer. IL-17A aggravates the development of gastric cancer, leading to a poor prognosis [
The study was authorized by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Nanjing, China, 2017-SRFA-064). A total of 15 NSCLC patients with the mean age of 61 (35–72) years who were treated in the First Affiliated Hospital of Nanjing Medical University between 2017 and 2018 were recruited, including 8 male and 7 female cases. Among them, there were 13 cases of lung adenocarcinoma and 2 of lung squamous cell carcinoma. All patients were excluded chronic obstructive pulmonary disease (COPD). NSCLC specimens and paracancerous ones were collected for use. According to the latest version of the Union for International Cancer Control (UICC) TNM Classification of Lung Cancer, there were 2 cases of stage I, 12 of stage II, and 1 of stage III. All recruited subjects did not have history of surgery, chemotherapy, radiotherapy and immunosuppressive therapy.
Fresh NSCLC and paracancerous tissues specimens were cut into small pieces and digested in RPMI-1640 medium containing 50 units/ml DNase I, 100 μg/ml hyaluronidase, 1 mg/ml Collagenase Type IV and 2 μM
Total RNA was isolated from TILs using TRIzol (Life Technologies, Foster City, CA, United States), and then reversely transcribed to cDNA with Prime Script RT Master Mix (Takara, Otsu, Japan) according to the manufacturer’s instructions. Relative levels were measured using the SYBR Green Master Mix on the 7500 Real-Time PCR system (Applied Biosystems; Life Technologies, Grand Island, NY, United States). IL-17 mRNA primers were designed by primer premier 5.0 software. The primer sequences were: IL-17, 5′-ACCAATCCCAAAAGGTCCTC-3′ (forward) and 5′-GGGGACAGAGTTCATGTGGT-3′ (reverse); glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-GAAGGTCGGTGTGAACGGA-3′ (forward) and 5′-GTTAGTGGGGTCTCGCTCCT-3′ (reverse). Relative levels were calculated by the 2−ΔΔCt method and normalized to that of GAPDH.
TILs were washed with phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS). Aliquots of single cell suspensions (1×106) were incubated with fluorophore-conjugated monoclonal antibodies for cell surface staining at room temperature in the dark for 20 min (Alexa Fluor 700 anti-human CD3 antibody, Alexa Fluor 750 anti-human CD45 antibody, PE anti-human CD4 antibody, FITC anti-human CD8 antibody, and/or BV421 anti-human TCRγδ antibody; all from Biolegend, San Jose, CA, United States). For intracellular staining, cells were cultured in RPMI-1640 medium containing 10% FBS, 50 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin, and 1 μg/ml brefeldin A (Biogems, Rocky Hill, NJ, United States) by for 5 h at 37°C. Cells were then washed with PBS containing 1% FBS, fixed, permeabilized using Intracellular staining perm wash buffer kit (Biolegend, San Jose, CA, United States) according to the manufacturer’s protocol, and stained with APC anti-IL-17A antibody (Biolegend, San Jose, CA, United States). Cells without staining were used as control. Th17 cells were defined as CD3+CD4+IL-17+ cells. Tc17 cells were defined as CD3+CD8+IL-17+ cells and γδT17 cells were defined as CD3+TCRγδ+IL-17+ cells. Fluorescence signals were collected on a FACS Aria II (BD Biosciences, San Jose, CA, United States) and analyzed with FlowJo software (Tree Star, Ashland, OR, United States). Absolute value of positive cells in 1 × 106 cells was calculated based on the positive percentage.
The levels of serum CYFRA 21-1 and CEA were detected by Roche Cobas E602 automatic electrochemiluminescence immunoanalyzer (ROCHE, Basel, Switzerland) in Department of Laboratory Medicine of the First Affiliated Hospital of Nanjing Medical University. The normal reference value range of CYFRA 21-1 is <3.3 ng/ml and that of CEA is <5 ng/ml.
Statistical analysis was performed using SPSS (Statistical Package for the Social Science) 20.0 statistical software (IBM Corp, Armonk, NY, United States). Pearson test was used to test whether the data come from a Gaussian distribution. Differences between the two groups were evaluated using the Student’s
The mRNA levels of IL-17A in TILs of NSCLC and paracancerous specimens were measured by qRT-PCR, which was significantly upregulated (2.208 ± 0.355 fold) in the former (
Expression level of IL-17A in TILs of NSCLC (T) and paracancerous specimens (N).
We then detected the expression level of IL-17A in CD3+ T cell subsets. IL-17 was expressed in infiltrated CD3+CD4+ cells, CD3+CD8+ cells and γδT cells of both NSCLC and paracancerous tissues (
γδT cells are the main source of IL-17A in NSCLC tissues. The positive rate of IL-17A+ cells in CD3+CD4+ cells
We further analyzed the correlation between IL-17+ γδT cells in TILs of NSCLC and clinicopathological features of NSCLC patients. As shown in
Correlation of different IL-17A+ cell subsets with the clinicopathological characteristics of NSCLC.
Features | N | Subsets ( |
|||||
---|---|---|---|---|---|---|---|
Th17 (Mean ± SEM %) |
|
Tc17 (Mean ± SEM %) |
|
γδT17 (Mean ± SEM %) |
|
||
Gender | |||||||
Male | 8 | 7.18 ± 1.18 | 0.51 | 6.24 ± 0.71 | 0.87 | 10.02 ± 1.56 | 0.16 |
Female | 7 | 8.56 ± 1.64 | 6.46 ± 1.08 | 13.06 ± 1.28 | |||
Age | |||||||
<61 | 6 | 6.10 ± 1.40 | 0.61 | 5.86 ± 1.01 | 0.55 | 11.48 ± 2.29 | 0.97 |
≥61 | 9 | 7.30 ± 1.47 | 6.69 ± 0.86 | 11.41 ± 1.07 | |||
Smoking | |||||||
Yes | 5 | 8.73 ± 2.45 | 0.59 | 6.00 ± 1.00 | 0.71 | 10.44 ± 2.39 | 0.52 |
No | 10 | 7.39 ± 1.19 | 6.54 ± 0.85 | 11.94 ± 1.14 | |||
Tumor size (cm) | |||||||
<4 | 8 | 7.02 ± 1.41 | 0.89 | 6.96 ± 0.84 | 0.55 | 12.52 ± 1.41 | 0.30 |
≥4 | 7 | 7.29 ± 1.31 | 6.18 ± 0.95 | 10.21 ± 1.6 | |||
Lymphatic metastasis | |||||||
Yes | 7 | 8.89 ± 1.33 | 0.19 | 7.20 ± 1.11 | 0.23 | 14.77 ± 1.04 | <0.001*** |
No | 8 | 6.20 ± 1.39 | 5.62 ± 0.67 | 8.52 ± 0.92 | |||
CYFRA 21-1 (ng/ml) | |||||||
<3.3 | 8 | 5.423 ± 0.79 | 0.06 | 5.639 ± 0.75 | 0.24 | 8.91 ± 0.91 | <0.01** |
>3.3 | 7 | 8.79 ± 1.47 | 7.18 ± 1.05 | 14.40 ± 1.39 | |||
CEA (ng/ml) | |||||||
≤5 | 9 | 7.66 ± 1.20 | 0.79 | 6.77 ± 0.86 | 0.45 | 11.01 ± 0.97 | 0.64 |
>5 | 6 | 8.25 ± 1.91 | 5.74 ± 0.99 | 12.08 ± 2.36 |
The correlation between Th17 cells, Tc17 cells, γδT17 cells in NSCLC specimens and clinical features of NSCLC patients.
In the tumor microenvironment, the inflammatory responsive involving both inflammatory cells and inflammatory molecules remarkably influences chronic inflammation and tumor development. IL-17A is a powerful proinflammatory factor, which used to be considered as the product of Th17 cells, a subset of CD4+αβT cells. The secretion of IL-17A requires antigen selection and activation in the thymus, which is inconsistent with the large-scale production of IL-17A in the early stage of inflammatory response. A growing number of studies have later found that IL-17A can be produced by various types of innate immune cells like γδT cells and natural killer (NK) cells. Notably, γδT cells have been confirmed as the most important source of IL-17A in the early phase of immune response [
At present, the role of IL-17A in inflammatory response is controversial. γδT17 cells contribute to enhance the immunity to fight against infections of
Howevever, the correlation between IL-17A and NSCLC has not been widely explored, and IL-17A level is usually measured in peripheral blood. We believed that IL-17A level in tumor specimens is more realistic to reflect the infiltration of inflammation in tumor sites, which is beneficial to identify the correlation between the microenvironment of NSCLC and inflammatory response. Cui K, et al. [
In the present study, IL-17A was significantly upregulated in TILs of NSCLC specimens and the main source of it was γδT cells. Moreover, CYFRA 21-1 is a prominent marker for NSCLC with the outstanding sensitivity and specificity [
In summary, IL-17A was highly expressed in TILs of NSCLC and mainly from γδT cells, which promoted the development of NSCLC. IL-17A and γδT17 cells may be a promising target for immunoassay and immunotherapy of NSCLC. Due to the difficulty of obtaining fresh tissues, our sample size was small. Limited by this reason, our conclusions should be further validated in large-scale studies. In addition, the underlying mechanisms of IL-17A in mediating the microenvironment of NSCLC need to be explored in the future.
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.
Conception and design: RX, TW, and SJ. Administrative support: TW and SJ. Provision of study materials or patients: SJ. Collection and assembly of data: RX, WS, and XK. Data analysis and interpretation: RX, SL, and XF. Manuscript writing and final approval of manuscript: all authors.
This study was supported by Natural Science Foundation of China under grant (No. 81772779) and “The Six Top Talent Project” of Jiangsu Province (No. 2015-WSN-034); and Jiangsu Province’s Key Provincial Talents Program (No. ZDRCA2016003), and Key Laboratory for Medicine of Jiangsu Province of China, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ZDXKB2016005).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Thanks for the technical support from National Key Clinical Department of Laboratory Medicine and Department of Respiratory and Critical Care Medicine of Jiangsu Province Hospital.
The authors have completed the TREND reporting checklist. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.