Clinical Study Of The Relationship Between Chemokine Receptor CXCR4and CCR7and Biological Behavior In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma is one of the most common malignant tumors worldwide, and its incidence is very high in China. Surgical resection is the primary treatment but the effect of the surgical resection is much far away from supposed to be. The overall5-year survival rate is only30%-50%. Clinically, it appears that lymph node metastasis of esophageal cancer may occur early, metastatic spread constitutes the primary source of morbidity and mortality, and the prognosis of patients with metastasizing esophageal cancer leaves much to be desired. Therefore, a thorough understanding of the lymph node metastasis is likely to be crucial to developing effective new therapies for esophageal cancer.Tumor metastasis is one of the most important biological behaviors of the tumor, which involves a series of complex processes with many factors. But the mechanism of metastasis is still unclear. Tumor metastasis results from a non-random process, but a selective process——Organ specificity. In1889Paget developed the theory of "seed and soil", and He hypothesized that certain tumor cells (seeds) colonize selectively distant organs (soil) with a favorable environment facilitating survival of tumor cells. Then in1928, Ewing explained metastases by the "anatomical or mechanical" theories such as tumor cell trapping or lodgement of tumor emboli into organ vascular bed. However, in the study of Tumor metastasis a new theory——the "homing" theory appeared. The theory holds that different organs have special abilities to arrest or attract through chemotactic factors specific types of cancer cells.In recent years, the research of chemokine receptor-ligand signaling axis provided compelling support for the chemoattraction part of the homing theory. Chemokines are a large family of small secreted proteins associated with the leukocyte trafficking during host defense and pathological immune responses. Chemokines are generally8～15kDa in size and contain70～125amino acids. The human chemokine system currently includes more than50different chemokines and20different chemokine receptors. They are classified into which are grouped into four families (C, CC, CXC, and CX3C) based on the spacing of key cysteine residues near the N terminus of these proteins. Chemokines act through both specific and shared receptors that all belong to the superfamily of G protein-coupled receptors (GPCRs) with seven-transmembrane domains. Chemokines and the specific receptors on cell surface may participate in a variety of physiological and pathological processes, such as cell growth, development, differentiation, apoptosis, tissue injury, tumor growth and metastasis. In2001, Muller A and the colleagues first demonstrated a potential mechanism for site-specific metastasis which is related to expression of chemokines and their receptors. They reported that the chemokine receptors CXCR4and CCR7are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1a and CCL21/6Ckine exhibited peak levels of expression in organs representing the first destinations of breast cancer metastasis. Their findings indicated that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.While nowadays the reports on the role of the chemokine-receptor axis in metastasis of esophageal cancer were very rare. This study was to investigate the expression of chemokine receptor CXCR4and its relationship to prognosis in esophageal cancer after esophagectomy by immunohistochemistry. And then this study was to investigate the relationship between CCR7expression and lymph node metastasis of esophageal cancer by RT-PCR, immunohistochemistry, and western blot assays and explore the mechanism of lymph node metastasis of esophageal cancer.Objective The incidence of lymph node metastasis of esophageal squamous cell carcinoma (ESCC) was shown to be positive correlated with tumor chemotactic migration and lymphangiogenesis. This study was to to investigate the expression of chemokine receptor CXCR4and its relationship to prognosis in ESCC after esophagectomy, and investigate the relationship between lymph node metastatic potentiality of ESCC and expression of CCR7and VEGF-C.MethodFrom June2003to June2006, we analyzed retrospectively the mid-thoracic ESCC patients who underwent complete resection(R0resection) in Provincial Hospital Affiliated to Shandong University with intact clinical information. The specimens consisted of143male and41female patients aged34-82years (average56.8), including132patients who underwent Ivor-Lewis esophagectomy and other52patients underwent esophagectomy through the left chest with esophagogastrostomy above the aortic arch. Preoperative radiotherapy or chemotherapy was not taken to all cases. The samples of cancer tissues, lymph nodes and normal esophageal tissues from60patients with mid-thoracic ESCC, who underwent complete resection in Provincial Hospital Affiliated to Shandong University from April2010to December2010. The expression of CXCR4was detected by immunohistochemistry. According to the clinicopathologic factors, the difference of CXCR4expression was compared by χ2test. Kaplan-meier method was performed to calculate the survival rate, Cox regression multivariate analysis was performed to determine independent prognostic factors.60samples were examined by RT-PCR and Western blot assays to detect CXCR4expression, χ2test was used to compare the difference of CXCR4expression to determine the relationship between invasion and metastasis of ESCC. And60samples were examined by RT-PCR, immunohistochemistry and Western blot assays to detect CCR7and VEGF-C expression. Monoclonal antibody D2-40was used to assessment of lymphatic vessel density (LVD). The SPSS16.0software package was used for statistical analysis. According to the clinicopathologic factors, the difference of CCR7and VEGF-C expression was compared by χ2test, and the difference of LVD was compared by u-test. Spearman's rank correlation analysis was used to determine the relationship between CCR7and VEGF-C expression. Logistic regression analysis was performed to determine the independent risk factors of influencing lymph node metastatic potentiality of ESCC.Results1. The expression rate of CXCR4in I stage, II stage and III stage was separately25.0%,69.6%and82.3%, the difference of CXCR4expression in I stage,Ⅱ stage and Ⅲstage was statistically significant (χ2=12.219, P=0.002). The CXCR4expression rate in, T2stage and T3stage was separately33.3%,64.7%and78.5%, the difference of CXCR4expression in T1stage, T2stage and T3stage was statistically significant (χ2=12.713, P=0.002). The positive expression of CXCR4in patients with lymph node metastasis was significantly higher than those without metastasis (χ2=8.600, P=0.003). The5-year survival rate in patients with CXCR4positive expression was significantly lower than those without CXCR4positive expression (P=0.000); the5-year survival rate in T2stage, NO stage, N1stage patients with CXCR4positive expression was significantly lower than those without CXCR4positive expression (P<0.05), but the5-year survival rate in T2stage patients with CXCR4positive expression was not significantly lower than those without CXCR4positive expression (P=0.075). The result of Cox analysis demonstrated that tumor invasion, lymph node metastasis and CXCR4positive expression were independent prognostic factors. CXCR4mRNA expression was observed in44(73.3%) esophageal cancer tissues by RT-PCR assay; CXCR4protein expression was observed in42(70.0%) esophageal cancer tissues by Western blot, respectively. CXCR4expression was positive related to tumor invasion and lymph node metastasis of ESCC.2. CCR7mRNA and VEGF-C mRNA expression was observed in46(76.7%) and33(55.0%) ESCC tissues by RT-PCR assay; CCR7protein and VEGF-C protein expression was observed in43(71.7%) and29(48.3%) ESCC tissues by immunohistochemistry; and CCR7protein and VEGF-C protein expression was also observed in44(73.3%) and31(51.6%) ESCC tissues by western blot, respectively. Each of CCR7and VEGF-C expression was positive related to lymph node metastasis of ESCC (P<0.05), furthermore co-expression of CCR7and VEGF-C expression was more significantly related to lymph node metastasis of ESCC (P<0.05). In ESCC tissues, LVD was higher than that in normal esophageal tissues (P<0.001); and LVD in ESCC was positively related to CCR7expression, VEGF-C expression and lymph node metastasis. The expression rate of CCR7mRNA and VEGF-C mRNA in metastatic lymph nodes were86.2%and69.0%, which were higher than that in non-metastatic lymph nodes(22.6%and19.3%). Obvious expression of CCR7and VEGF-C protein was found in no normal esophageal tissuse. Spearman's rank correlation analysis showed that CCR7expression was positive correlated with VEGF-C expression. Logistic regression analysis revealed that CCR7mRNA expression(OR=12.044, P=0.012), VEGF-C mRNA expression(OR=5.976, P=0.015) and tumor invasion(OR=0.110, P=0.012) were independent risk factors of lymph node metastasis of ESCC.ConclusionsThe patients with CXCR4expression have poor prognostic. CXCR4expression was molecular biomarker to judge the prognostic of patients with ESCC after esophagectomy.CCR7and VEGF-C were highly expressed in ESCC and the metastatic lymph nodes, with significant correlation with lymph node metastasis of ESCC. The co-expression of CCR7and VEGF-C might promote lymphatic metastasis of ESCC and might be a clinical predictor of lymph node metastasis of patients with ESCC.