| Background and aimLung cancer is one of the more common form cancer worldwide, and its incidence increases with the change of environmental pollution and human lifestyles.Non-small cell lung cancer is the common type of lung cancer, accounting for about 85% of the population of lung cancer. Esophageal cancer is one of the high mortality rate cancer, the death rate is among the first eight cause of death, and the 5-year survival after surgery is only 20~30 %. Despite With imaging technology, and advances in surgery, radiotherapy and chemotherapy progress, the survival rate has not significantly be improved, which makes the treatment of such cancers face greater challenges.In human body, the function of the immune systemis to recognize and kill tumor cells, however, the immune system has been significantly inhibited, a large number of innate and adaptive immune cells involved in the immune suppression. In Tumor immunosuppressive microenvironment, tumor cells and tumor stroma secrete chemokines and cytokines, recruit Tregs cells, bone marrow-derived suppressor cells(myeloid-derived suppressor cells, MDSCs), Th17 cells and B-Reg cells(regulatory B cells) to the tumor tissue, formating immunosuppressive microenvironment to promote tumor progression. Studies have shown that, in many types of primary or metastatic tumors tissues, such as ovarian cancer, breast cancer, liver cancer, lung cancer, oral squamous cell carcinoma, prostate cancer, squamous cell carcinoma, etc., a large number of Tregs cell infiltration can be found, and Tregs cells is closely related to the prognosis of cancer, a large number of Tregs cell infiltration sugges that the prognosis is poor.A number of studies have showed that chemokines play an important role in immune cell migration movement. CCL20 chemokine specific binds to receptor CCR6 and participates in Treg cells migrating to the tumor site, so in a variety of cancers, such as colorectal cancer and squamous cell carcinoma of the larynx, CCL20 play an important role in rogression and metastasis of cancer. However, the role of chemokines CCL20 factor in non-small cell lung cancer has not yet been revealed. CXCL8 is an important multifunctional cytokine, through autocrine and paracrine pathway, binds to its receptor CXCR1 and CXCR2, play a very important role in the development process of breast cancer, lung cancer, colorectal cancer and melanoma.The study of the role of CXCL8 in the process of developing esophageal cancer is less. IL-32 is an important pro-inflammatory cytokines, is highly expressed in cancer tissues, activating NF-k B pathway by stimulating release of cytokines such as CXCL8, and promote the development of cancer. In a variety cancers, CXCL8 can make Tregs cell migrating to tumor tissue, whether IL-32 can act on CXCL8 to promote the development of esophageal cancer is less researched. The present study was to investigate the mechanisms of recruiting Tregs by chemokine CCL20 and the cytokine IL-32 in small cell lung cancer and esophageal squamous cell carcinoma cells, in order to provide new therapeutic methods and ideas.Part I The expression pattern of chemokine CCL20 in NSCLC and its role in the recruiting of Treg cells to tumor site 1 Background and objectRevealing the mechanism of CCL20 recruiting Treg cells to NSCLC tumortissue. 2 Methods2.1 RT-PCR was used to detect the expressions of CD4/CCL20/Foxp3/IL-10 in the NSCLC tumor tissues or the matched adjacent normal samples. Also, we analyzed the significance of CCL20 in the patients stage and survival rate;2.2 We used FACS to reveal the Treg cell fraction in TIL and PBMC, and the expression of CCL20 receptor, CCR6, in the Treg cells both from TIL and PBMC;IHC was used to analyze the expression of CCL20 in protein level and detect its main source;We used ELISA to analyze the expression of CCL20 with or without docetaxel treatment. 3 Results3.1 According to the RT-PCR and IHC, we found that chemokine CCL20 was highly expressed in the NSCLC tumor site compared with the matched adjacent normal samples;3.2 In the NSCLC tumor samples, the expression of chemokine CCL20 was positively correlated with Treg cell-markers, CD4 and Foxp3, and CCL20 was also positively associated with pro-inflammatory cytokine IL-10;3.3 The FACS data showed that the fraction of Treg cells in TIL from NSCLC tissues was higher compared with the matched PBMC from patients blood;3.4The expression of chemokine CCL20 in the NSCLC tumor sites was an indicator of patients disease and prognosis, with both negative correlations;3.5 Docetaxel treatment decreased the expression of CCL20 in the A549 cell line. 4 ConclusionsChemokine CCL20 co-operated with its receptor CCR6 could enhance the ability of Treg cell migrating to NSCLC tumor sites. Considered the role of docetaxel in decreasing the expression of CCL20 in A549 cell line, docetaxel may have the ability to reduce the percent of Treg cell migrating to tumor sites.Part II The expression pattern of cytokine IL-32 in esophageal cancer and its immunological function 1 Background and PurposeThis aims to explore the related mechanism of IL-32 recruiting Tregs cell in esophageal cancer tissue, in order to provide new method and train of thought for the treatment of esophageal cancer. 2 Methods2.1 The RT-PCR was used to detect the expression of IL-32, Foxp3, CXCL8 in esophageal tumor and matched adjacent tissues or in the cell lines in gene level.2.2 The IHC was used to detect the expression of IL-32 in the esophageal cancer tissues in protein level.2.3 si RNA technology was used to knockdown the expression of IL-32 in TE1 cell line, and further testing CXCL8 expression level, and detecting its effect on migration of Treg cells.2.4 Using flow cytometry, we analyzed the percent of Treg cells in TILs that isolated from the esophageal cancer tissues as well as in the PBMC that obtained from patients blood.2.5 Using SPSS17.0 software, we analyzed the correlations among the expression of IL-32 with the clinical data and postoperative survival time that from the patients. 3 Results3.1 According to the RT-PCR results, we found that the expression of IL-32 was higher in esophageal cancer tissues compared with the matched adjacent tissues. And the expression of IL-32 varied from the cell lines, it nearly had no expression in the esophageal normal cell line-Het-1?and the esophageal cancer cell lines, KYSE450 and KYSE70, while had highest expression in TE1 cells.3.2 RT-PCR results showed that the expression of IL-32 was positively correlated with and chemokines CXCL8. And in the esophageal cancer tissues, the expression level of CXCL8 was positively correlated with Treg cell markers, CD4 and Foxp33.3 The expression of CXCL8 was lowered after si RNA gene knockout in the cell line TE1, both in genetic level and migration of Treg cells.3.4 Flow cytometry data showed that the percent of Treg cells in TIL was much higher than in PBMC. And in the esophageal cancer tissues, the expression level of IL-32 was correlated with Treg cell markers, CD4 and Foxp3.3.5 According to the clinical analysis data, we found that the expression of IL-32 was negatively related to the level of clinical stage, differentiation, survival time after operation and lymph node metastases in patients with esophageal cancer patients. 4 SummaryIL-32, as a pro-inflammatory cytokine, involved in the formation of esophageal cancer local microenvironment, by promoting the secret of chemokine CXCL8 which elevated the number of Treg cells infiltrated to the tumor site. |
PDF Full Text Request