Increased IL-17-producing Cells Correlate With Poor Survival And Lymphangiogenesis In NSCLC Patients

BackgroundNon-small cell lung caner (NSCLC) is the leading cause of cancer death worldwide. The outcome of patients with NSCLC remains poor and the overall 5-year survival rate is still around 15% even with combined treatment including surgery, chemotherapy, radiotherapy and targeted therapy. Inflammation, angiogenesis and lymphangiogenesis play an important role in tumor progression. Interleukin 17 is a pro-inflammatory cytokine mainly produced by activated T cells. IL-17-producing CD4+ T helper cells, termed Th17 cells, have potent pro-inflammatory properties and play an active role in inflammation and cancers. Recently, accumulating evidence has shown that IL-17-positive cells are frequently present in multiple inflammation-associated cancers, including prostate cancer, colorectal cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, as well as NSCLC. Most of the literature reports show that IL-17 promotes angiogenesis in tumor models and IL-17 expression correlates well with the numbers of blood vessels in human ovarian cancer, hepatocellular carcinoma and NSCLC; although some reports indicate that IL-17 may protect against tumors by promoting immune system-mediated tumor rejection. However, the relationship between the density of IL-17-producing cells and the prognosis for the patient with NSCLC has not been evaluated.The proinflammatory function and intracellular signaling pathway of IL-17 is similar to those of IL-1 and Toll receptors. IL-17 has several biologic activities including induction of IL-6, IL-8, TNF-α, IL-1β, and VEGF. So we tested the possibility that IL-17 might have a deleterious role in patients with NSCLC by promoting lymphangiogenesis via enhancing the expression of VEGF-C, involved in lymphangiogenesis.ObjectiveThe aim of this study is to determine the prognostic significance of IL-17 in NSCLC patients and to examine the correlation between IL-17 expression and lymphatic vessel density in NSCLC tissues. Next we tested the possibility that IL-17 might have a deleterious role in patients with NSCLC by promoting lymphangiogenesis via enhancing the expression of VEGF-C, involved in lymphangiogenesis.Methods1. In order to identify the cell type that was producing IL-17, we stained consecutive sections for the T cell markers CD4, CD8 and the macrophage marker CD68. The expression of IL-17 was measured by immunohistochemistry in 52 paraffin-embedded tissues with non-small cell lung cancer. Theχ2 test was used to analyze the correlation between IL-17 expression and clinical parameters. The Kaplan-Meier method, univariate and multivariate regression analysis was used to analyze the correlation between IL-17 expression and overall survival and disease-free survival.2. Theχ2 and T test was used to analyze the correlation between IL-17 expression and lymphatic vessel density (LVD) and VEGF-C expression in 36 paraffin-embedded tissues with NSCLC.3. MTT assay was used to determine whether IL-17 cytokine affects LEC cells growth. Tranwell experiment and tube formation assay was used to determine whether LLC/IL-17 CM (condition media) affects cell migration and tumor formation of LEC cells, respectively. Flow cytometry test was used to test the the expression of IL-17R on LLC and LEC.4. IL-17 was used to stimulate Lewis lung caicinoma (LLC) cells and the expression of cytoplasmic and secreted VEGF-C, and mRNA were determined by ELISA and quantitative RT-PCR assay. To assess the role of ERK 1/2 activation in IL-17-mediated VEGF-C induction, A549 cells were stimulated with IL-17 in the presence or absence of PD98059, a specific inhibitor of ERK 1/2. IL-17-induced VEGF-C expression was tested when ERK 1/2 activity was inhibited of by PD98059Results1. Cells which were positive for CD4 or CD8 occurred in the same area of the tumor as those cells that produced IL-17 and these cells exhibited the same morphological characteristics as IL-17 positive cells. IL-17 positive cells that exhibited polymorphonuclear neurophil characteristics were also observed. High expression of IL-17 was observed in 25 of 52 lung cancer patients and was associated with smoking status, TNM stage, overall survival and disease-free survival. Univariate and multivariate analysis showed that IL-17 was an independent prognostic factor for overall survival and disease-free survival.2. In recent study of 36 NSCLC patients, the X2 test showed a significant correlation between expression of IL-17 and VEGF-C. (P<0.01). A similar association was observed between VEGF-C expression and LVD (p<0.01).3. IL-17 had no direct effect on the proliferation, chemotactic response or endothelial cord formation of LECs. The LLC culture supernatant caused an increase of chemotaxis and endothelial cord formation of LECs than IL-17(P<0.05). Moreover, greater increased chemotaxis and endothelial cord formation of LECs was observed in response to CM from LLC/IL-17 (P<0.05). By flow cytometry test we found that LLC cells but not LECs expressed IL-17R. These results indicated that IL-17 had lymphangiogenic activity by inducing lymphangiogenic cytokines production of LLC.4. We found that IL-17 increased the expression of VEGF-C mRNA and protein levels in LLC. Stimulation of LLC by IL-17 with 0.1, 1, 10, 100, 500 ng/ml increased the VEGF-C mRNA expression after 6 h culturing. The VEGF-C mRNA level was elevated with a peak in the 10 ng/ml IL-17 treatment. The VEGF-C mRNA expression of LLC unstimulated, stimulated by IL-17 (10 ng/ml) was 1 and 1.40±0.17 after 6h culturing. VEGF-C protein production of LLC unstimulate, stimulated by IL-17 (10 ng/ml) was 203.0±13.1 and 277.3±14.0 ng/ml after 48 h culturing. IL-17 mediated expression of VEGF-C could be also observed in other IL-17R+ human lung cell lines, such as A549. The addition of IL-17 to A549 cultures activated ERK 1/2. Inhibition of ERK 1/2 activity by PD98059 significantly inhibited IL-17-induced VEGF-C expression at the RNA levels.Conclusions1. IL-17 was an independent prognostic factor for overall survival and disease-free survival in NSCLC. IL-17 positive cells observed were mainly neurophil and T cells.2. X2 test showed a significant correlation between expression of IL-17 and VEGF-C and LVD. IL-17 can be implicating as a lymphangiogenic factor which promote lymphangiogenesis via up-regulation of VEGF-C of lung cancer cells.3. IL-17 had no direct effect on the proliferation, chemotactic response or endothelial cord formation of LECs, but greater increased chemotaxis and endothelial cord formation of LECs was observed in response to CM from LLC/IL-17 compared with LLC culture supernatant. By flow cytometry test we found that LLC cells but not LECs expressed IL-17R.4. IL-17 increased the expression of VEGF-C mRNA and protein levels in LLC. Inhibition of ERK 1/2 activity by PD98059 significantly inhibited IL-17-induced VEGF-C expression at the RNA levels.