Conceived and designed the experiments: UI OMS AP GZ OW. Performed the experiments: SK KB ZS HW JD. Analyzed the data: JD GA FV OW. Wrote the paper: OW.
The authors have declared that no competing interests exist.
Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.
A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.
CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.
Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.
Primary carcinoma of the lung is the second most frequent (12%) cancer worldwide, and is the leading cause of cancer related death. NSCLC (mainly lung adenocarcinoma) accounts for nearly 80% of cases. Lung cancer is linked to a long history of smoking and to its accompanying chronic inflammatory response
Distinct cytokine and chemokine/chemokine receptors characterize specific types of immune responses
The chemokine/chemokine receptor pair CCL20/CCR6 is a key player in lung immunity
The expression, regulation and function of CCL20/CCR6/IL-17 in NSCLC have not been characterized thus far. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.
Fresh human lung and tumor specimens were obtained from patients (n = 20) undergoing complete resection of early stage NSCLC (clinical stage IA-IIB) who had not received preoperative chemotherapy or radiotherapy to exclude confounding effects. Histological sections were prepared from these samples and an experienced pathologist (GA) confirmed the histopathological diagnosis. These tissues were used for the various experiments described in this manuscript. In order to assess the correlation between CCL20/CCR6 expression and lung adenocarcinoma disease progression, we additionally collected 49 paraffin-embedded tissue sections of lung adenocarcinoma tumors (clinical stage IA-IIB) that were removed from patients in our department. The study period was January, 2000 to September, 2010. Patients did not receive preoperative chemotherapy or radiotherapy to exclude confounding effects. All patients underwent an extensive sampling of mediastinal lymph nodes. An experienced pathologist (GA) reassessed the slides to re-confirm the diagnosis. Clinical data (survival, time to disease recurrence and pathological staging) of these patients was reviewed. The Hadassah Hospital Ethics Committee approved the human component of the study. A written informed consent was obtained from all participants involved in this research.
Assessment of CCR6 expression in lung adenocarcinoma and correlation analysis to pathologic stage of disease were also done using the Biomax tissue array: BC041115, which is a lung carcinoma and normal tissue array (Biomax US. 1100 Taft St., Rockville, MD 20850, USA).
Antigen retrieval was performed in EDTA buffer for 20 minutes in microwave. Sections were stained with the anti-human CCR6 monoclonal antibody (R&D Systems, Minneapolis, MN 55413, USA) - concentration 10 mg/ml, or with anti-human CCL20 polyclonal antibody (PeproTech EC, London, UK), - concentration of 20 mg/ml. Next, the sections stained for CCL20 were incubated with diluted 1∶1000 biotinylated goat-antirabbit antibody (Jackson ImmunoResearch), for 30 minutes and thereafter with horseradish peroxidase - conjugated streptavidin (Zymed Laboratories, San Francisco, CA,USA) for 30 minutes. The sections stained for CCR6 or CCL20 were incubated with secondary anti-mouse horseradish peroxidase-conjugated antibody (DakoCytomation, Glostrup, Denmark) for 30 minutes at room temperature. 3-amino-9-ethylcarbazole (AEC) was used for color development, and sections were counterstained with hematoxylin. Negative control sections were stained with either no primary antibody (PBS) or with isotype-matched control antibody.
Two experienced pathologists blindly assessed tissue sections for the percentage of tumor cells staining positive for CCL20 and CCR6. Samples were divided into four categories: 1. 0–25% of tumor cells stained positive, 2. 25–50% of tumor cells stained positive, 3. 50–75% of tumor cells stained positive, 4. 75–100% of tumor cells stained positive. High expression of the chemokine or receptor was defined if more than 50% of tumor cells stained positive.
Analysis of the Biomax lung cancer tissue array was done as follows: CCR6 staining intensity was scored from 0 to 3 (0 – no staining, 1 – week intensity, 2 – medium intensity and 3 – high intensity); the percentage of CCR6 positive cells (four categories as described above) was also scored. For each sample, we calculated a CCR6 staining index: CCR6 index = (CCR6 Staining intensity * Percentage of CCR6 positive tumor cells)/100.
Tumor and lung tissue homogenates were prepared on ice from weighed tissue samples. CCL20 levels in these homogenates were measured. Levels of CCL20 in the supernatant of NSCLC-derived cell lines (L3, L4 and L549) were measured in the presence or absence of IL-17 (25, 100, 1,000 ng/ml). IL-17 levels were measured in the supernatant of tumor-derived immune cells and in the supernatant of lung-derived immune cells in the presence or absence of anti OKT3 antibody. ELISA assays were performed with Quantikine kits according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN 55413, USA).
Isolation of normal lung- and tumor tissue-derived lymphocytes was done as previously described
The A549 – cell line was purchased from the ATCC (Rockville, MD, USA). Two primary NSCLC cell lines were generated in our laboratory as previously described; the L3 line was generated from lung adenocarcinoma and the L4 line from large cell carcinoma
In order to ascertain that our NSCLC-derived cell lines, L3 and L4, differ from each other and from the A549 ATCC cell line, we performed fingerprinting of these cells as described by Silva et al.
We performed flow cytometry analysis of 1.5X105/ml of primary tumor-derived neoplastic cells (L3 and L4) and the NSCLC cell line A549. Cells were blocked with 1% human plasma for 15 minutes and then mixed with PE - conjugated anti-CCL20 monoclonal antibody clone 67310 or with monoclonal anti-CCR6 antibody, clone 53103.11 (R&D Systems, Inc., Minneapolis, MN 55413, USA) or with isotype control for 20. For CCL20 staining cells, the fixation and permeabilization method was performed prior to incubation with the antibody. Immunostained cells were analyzed by flow cytometry using the FACS Caliber Flow Cytometer (Becton Dickinson, Mountain View, CA, USA). Data were analyzed using the CellQuest software. (Version 3.3, Becton Dickinson).
We used the anti-CCR6 antibody clone 53103.11 in both flow cytometry and immunohistochemistry staining. To demonstrate the specificity of this clone for CCR6, we incubated fresh PBMC in the presence or absence of 20 ug/ml CCL20 for one hour and stained the cells for CCR6 or with isotype control antibody. Reduced CCR6 staining was observed in CCL20 treated cells; results are provided as supplementary material (
Western blot analysis of ERK and phosphorylated ERK was carried out as previously described and according to the manufacturer's recommendation
Agar base layer was prepared as follows: 45 ml of RPMI +12% FCS was mixed with 15 ml of RPMI X2 + 12% FCS and with 15 ml of 2.5% agar in double distilled water. Tumor cells were suspended in RPMI + 10% FCS. Cell suspension was mixed in a ratio of 1∶3 with the agar base solution. This mixture was then plated on top of a preformed solid agar base. CCL20, at the concentrations of 10 ng/ml, 50 ng/ml and 250 ng/ml, and the ERK inhibitor PD98059 at the concentration of 20 microliter/ml, were added to the mixture. Fourteen days later, the number of colonies was counted in ten different fields.
The following primers were used:
CCL20 – sense
CCR6 – sense
IL-17A – sense
IL-17A receptor – sense
b-actin – sense
Data are expressed as mean ± standard error (SE) and as absolute values. Continuous data with normal distribution were analyzed using t-test. Time to disease recurrence was estimated non-parametrically by the Kaplan-Meier method. Cox regression analysis was performed to define the relevance of high CCR6 expression as a disease stage independent marker for disease recurrence. A p value of 0.05 was considered significant. Statistical analyses were performed using the SPSS software (IBM).
The expression of CCL20 and CCR6 in fresh NSCLC samples was assessed by PCR and ELISA assays.
PCR signal for CCL20, CCR6 and beta-actin in three NSCLC tumor samples (A). CCL20 protein levels in NSCLC tumors and in tumor adjacent lung tissue (n = 3) (B). Representative immunohistochemistry staining for CCL20 and CCR6 in lung adenocarcinoma tissue sections. CCL20 - Low X10 (D) and high power X20 (E) magnification. CCR6 - Low power (X10) (F, G) and high power (X20) (H) magnification. Immune cell infiltrates located with in lung adenocarcinoma tumor stain positive for CCR6 (X40) (I). Negative control staining (X10) is shown (C). (*P<0.05)
Using immunohistochemistry, we defined the expression patterns of CCL20 and CCR6 in lung adenocarcinoma. Low X10 and high power X20 magnification of CCL20 staining is shown in
To study the clinical relevance of CCL20/CCR6 expression in NSCLC, we tested the expression of the chemokine and receptor in 49 lung adenocarcinoma tissue samples removed during surgery. Results were correlated with patient pathological stage of disease and with time of disease recurrence. Patients' characteristics are shown in
Kaplan-Meier analysis of disease free survival interval (months) according to disease stage. (A). Kaplan-Meier analysis of disease free survival interval (months) according to percentage of CCR6 positive cells. (B). CCR6 staining index for lung adenocarcinoma and squamous cell carcinoma according to disease stage is shown (C).
N = | 49 |
Age | 65.8 +/− 8.91 |
Gender | 25 female/24 male |
Stage I | 32 |
Stage II | 9 |
Stage III | 8 |
Follow up | |
Mean | 54 +/− 23.5 months |
Median | 52 months |
Range | 18–125 months |
CCL20 | 0–25% | 25–50% | 50–75% | 75–100% |
Stage I | 6 | 2 | 5 | 19 |
Stage II | 1 | 0 | 2 | 6 |
Stage III | 2 | 0 | 2 | 4 |
Total | 9 | 2 | 9 | 29 |
Samples were divided into four categories according to percent of tumor cells staining positive: 1. 0–25% of tumor cells stained positive, 2. 25–50% of tumor cells stained positive, 3. 50–75% of tumor cells stained positive, 4. 75–100% of tumor cells stained positive.
CCR6 | 0–25% | 25–50% | 50–75% | 75–100% |
Stage I | 19 | 7 | 3 | 3 |
Stage II | 9 | 0 | 0 | 0 |
Stage III | 6 | 0 | 0 | 2 |
Total | 34 | 7 | 3 | 5 |
Samples were divided into four categories according to percent of tumor cells staining positive: 1. 0–25% of tumor cells stained positive, 2. 25–50% of tumor cells stained positive, 3. 50–75% of tumor cells stained positive, 4. 75–100% of tumor cells stained positive.
To examine the correlation between CCR6 expression and NSCLC disease stage, we measured CCR6 staining index in a tumor tissue array enclosing lung adenocarcinoma samples homogenously spread among the different disease stages. The array included 52 samples of lung adenocarcinoma (19 samples of stage I disease, 16 samples of stage II disease, 16 samples of stage III disease and 1 sample of stage IV disease) and 41 samples of squamous cell carcinoma (20 samples of stage I disease, 6 samples of stage II disease and 17 samples of stage III disease). The average CCR6 staining index was 0.75 +/− 0.16 for stage I adenocarcinoma, 1.05 +/− 0.24 for stage II adenocarcinoma and 1.37 +/− 0.22 for stage III adenocarcinoma. The increased CCR6 staining index observed in stage III relative to stage I adenocarcinoma was statistically significant P = 0.03 (
We characterized the expression of CCL20/CCR6 in three NSCLC-derived cell lines (L3, L4 and A549). A low level of CCR6 expression was detected in all three-cell lines (
PCR signal for CCL20, CCR6 and beta-actin in L3, L4 and A549 cell lines (A). ELISA assay for CCL20 in the supernatant of L3, L4 and A549 cell lines (B). Flow Cytometry histogram analysis of CCR6 and CCL20 staining of L3, L4 and A549 cell lines, control antibody – purple, staining antibody – green line (C).
The effect of CCL20 stimulation on neoplastic cell proliferation was studied using colony assays. Stimulation of L3, L4 and A549 cells by increasing concentrations of CCL20 promoted colony formation in a dose-dependent fashion (
Colony formation by L3, L4 and A549 cells in response to stimulation with increasing concentrations of CCL20 (A, B, C). ERK phosphorylation in L3, L4 and A549 cells in response to CCL20 stimulation. Phosphorylated ERK upper panel and total ERK lower panel (D, E, F). (*P<0.05, **P<0.01)
Colony formation by L3, L4 and A549 cells in response to stimulation with increasing concentrations of CCL20 in the presence or absence of the ERK inhibitor PD98059 (A, B, C).
The expression of IL-17A and IL-17A receptor in NSCLC tissue samples in tumor adjacent lung tissue and in L3, L4 and A549 cell lines was tested. PCR signal for IL-17A was low in two samples and negative in an additional two samples of tumor adjacent lung tissue (
PCR signal for IL-17A, IL-17A receptor and b-actin in NSCLC tissue samples, in tumor adjacent lung tissue and in L3, L4 and A549 cell lines. Tumor adjacent lung tissue (A). NSCLC tumor sample (B). L3, L4 and A549 cell lines (C). Infiltrating immune cells were isolated from tumor tissue (TD-IC) and tumor adjacent lung tissue (LD-IC). Immune cells were incubated with or without anti CD3 antibody and production of IL-17 assessed by ELISA - two cases (D, E). Semi-quantitative PCR analysis for CCL20 in L3, L4 cells 549 cells in response to IL-17 and IL-22 stimulation (F, G, H). ELISA assays for CCL20 in the supernatant of L3, L4 cells 549 cells following stimulation with increasing concentrations of IL-17 (I, J, K).
Chemokine receptor expression by tumor cells enhances their survival, proliferation and metastatic potential. Both tumor cells and tumor stroma produce chemokines
To test in vitro the potential effects of CCL20/CCR6 interactions in NSCLC, three tumor cell lines were tested. L3, L4 and A549 cells expressed low levels of CCR6 and produced varying levels of CCL20. Stimulation of these lines with CCL20 induced colony formation in a dose-dependent manner. CCR6-dependent ERK phosphorylation mediated proliferation of colorectal cancer cells
Autocrine and paracrine chemokines/chemokine receptor interactions link chronic inflammation to malignancy
In conclusion, our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.
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The authors thank Dr Ido Dov Weiss for assistance in revision and reviewing of the manuscript. The authors are grateful to Ms Mery Clausen for editing the manuscript.