The Significance Of TSLP Expression In Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is the most popular head and neck carcinoma, which accounts for approximately5.6%of all malignant cancers. Recently, the survival of the OSCC patients has been significantly increased. The5-year survival rate reached60%. It’s well known that the occurrence of the oral squamous cell carcinoma is a process involved many factors, including radiation, tooth bad stimulation, alcohol, tobacco and micro-nutrient deficiency, etc. With the development of tumor immunology, the immune mechanisms in oral cancer pathology receive more and more attentions. The changes in the immune regulation and tumor specific antigen immune responses can cause loss of immune surveillance and help tumor initiation and progression through immune escape.TSLP (thymic stromal lymphopoietin), a similar cytokine as IL-7, was first reported by Friend in1994. It was isolated from thymic stromal cells of mice in culture supernatant. It plays an important role in regulating cell growth and differentiation of B lymphocytes, cell proliferation of T lymphocytes and activation of dendritic cells. TSLP expression can be found in many different tissues, including intestinal epithelia, stimulated gastric mucosa, esophageal mucosal epithelium, heart, liver and prostate, etc. It can also be produced by skin epithelium, stromal and muscle cells. According to the recent researches, TSLP can also be expressed in different kinds of tumor cells, including breast cancer, pancreas cancer, melanoma and lung cancer, etc. TSLP can promote tumor progression through increasing the Th2cell proliferation and differentiation. However, there are very few researches on the TSLP expression in OSCC. In this paper, we will study the TSLP expression and its relationship with tumor development in OSCC cells by several approaches.Part I:The clinical significance of TSLP expression in patients with squamous cell carcinomaOral squamous cell carcinoma (OSCC) is one of the most popular head and neck carcinoma. TSLP, a similar cytokine like IL-7, was first reported by Friend et al in1994. TSLP expression can be found in many different tissues, and promote tumor progression via different mechanisms. However, there are very little researches have been done on TSLP expression in OSCC patients. In this paper, we studied the TSLP expression and its relationship with clinicopathological parameters.Objectives and MethodsThirty tissue samples from OSCC patients with complete clinical data and10Oral mucosa tissues from healthy control were included in this study. We studied the TSLP mRNA and protein expression through the methods of and RT-PCR and Immunohistochemistry. The TSLP concentrations in peripheral blood were compared between OSCC patients and healthy control through ELISA. Its association with clinical endpoint was also analyzed.ResultsTo quantify the TSLP and TSLP-R expression, tumor cell lines were examined by Immunohistochemical staining. Their expression in OSCC tissue and para-carcinoma tissues are high, but no significant expression in epithelium of the normal tissues. The mean TSLP mRNA level is1.9185times higher than normal tissue (0.016516±0.00352vs.0.008609±0.00045). TSLP concentration in peripheral blood is26.16±13.37and is significantly higher than in healthy controls (3.35±3.31) with p value less than0.001. TSLP expression levels are not significantly related to the clinical stages, pathological classification, age, sex.ConclusionSignificantly higher TSLP expression in OSCC tissues and peripheral blood is possibly related to the development of the oral squamous cell carcinoma through its autocrine from tumor cells. It can promote tumor development though regulating the tumor cells and/or microenvironments of the lymph node cells. There was no significant relevance between expression of TSLP in OSCC and age, gender, pathology grade and tumor size.Part2:Interaction between TSLP and FOXP3Expressions in patients with OSCCThe immune regulation is one key factor of the OSCC progression. We have shown that TSLP and TSLP-R can be extensively expressed in both OSCC cells and infiltrating lymphocytes. TSLP can induce the Treg cells differentiation from CD4+CD25-to CD4+CD25+Treg cells, which can specially expressed FOXP3and FOXP3+Treg cells play an important role in n the cell division and proliferation. The high level of Tregs plays an important role in cancer progression and metastasis by suppressing tumor specific immune reaction. However, the mechanism for the increase in Fox3+Treg in OSCC microenviroment is unclear. In this experiment, we studied the interaction among FOXP3, TSLP in the immune regulation.Objectives and MethodsThirty tissue samples from OSCC patients with complete clinical data and10Oral mucosa tissues from healthy control were included in this study. By using immunohistochemical staining, we investigate the TSLP-R, FOXP3expression and the co-expression of both these two proteins in lymphocytes infiltrating in OSCC. We also examined the co-expression of these two markers by using immunohistochemical staining in lymphocytes in the metastatic lymph nodes to explore the TSLP’s role in the OSCC lymph node metastasis.ResultThere are a high amount of FOXP3-expressing lymphocytes in OSCC tissues. The mean percentage of cells with positive FOXP3expression was23.32%(SD=4.3%) in OSCC patients and the mean is18.24%(SD=4.8%) in para-tumors. The mean expression in FOXP3is statistically significant higher in OSCC tissue sample and para-tumor tissues than that in oral mucosa of healthy controls.There are a high amount of TSLP-R expressing lymphocytes in the infiltrating lymphocytes mingling around the nests of neoplastic OSCC cells.There is a considerable amount of infiltrating lymphocytes co-expressing TSLP-R and FOXP3mingling around the nests of neoplastic OSCC cells. We also found TSLP-R and FOXP3co-expression in the metastatic lymph nodes.Conclusion:TSLP can probably promote the maturation of the FOXP3+Treg cells in OSCC microenvironments, which can inhibit the immune reaction and promote OSCC progression and lymph node metastasis.Part3:Associations between TSLP and OSCC cell proliferationThe uncontrolled cell proliferation caused by different mechanisms is the important pathogenesis reason. In the first part of the experiment, we found that TSLP can work on tumor cells with combined wih TSLP-R through paracrine.or autoncrine method In the very early experiments, TSLP have been found to be able to promote tissue or immune cell proliferation in direct or indirect manners. In OSCC, does TSLP promote tumor development through stimulating cell proliferation? In this experiment, we studied the relationship between TSLP and cell proliferation.Objectives and MethodsOSCC Tcs8113cell lines were used in this part of the experiment. Cell cultures with TSLP, TSLP neutralizing antibody and negative controls were prepared separately. The TSLP effects were analyzed though comparing Tca8113cell proliferations at different time points by MTT method.ResultsDifferential cell proliferations are observed after24hours in these three groups. Cell proliferation in the group with TSLP is higher than that in control group and the group with TSLP neutralizing antibody. But this difference is not statistically significant. There is no significant difference between control group and the group with TSLP neutralizing antibody.But, there were significant increases in cell proliferation in both TSLP group and control group after48hours. However, the increase is more significant in TSLP group.The increases in cell proliferation are more significant after72hours. TSLP group is significantly higher than the other two groups. Control group is also significantly higher than that in the group with neutral antibodies.Conclusion: TSLP can promote OSCC progression through inducing OSCC cell proliferation, which may lead to the pathogenesis and development of OSCC.