Clinical Study Of The Relationship Between Chemokine Receptor CCR7 And CXCR4 And Organ Metastases In Lung Cancer

BackgroundTumor 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. In 1889 Paget 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 in 1928, 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 generally 8-15 kDa in size and contain 70～125 amino acids. The human chemokine system currently includes more than 50 different chemokines and 20 different 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. In 2001, 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 CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-la 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.Nowadays, lung cancer is one of the most serious malignant diseases threaten to people's health in the world. In China, lung cancer occurs at very high frequencies and has been the leading cause of tumor-specific death, and more than 80% are non-small cell lung cancer (NSCLC). Clinically, it appears that lymph node metastasis of lung cancer may occur early, and latter lung cancer can metastasize to distant specific target organs, such as brain, bone, liver and adrenal glands. Among them, brain metestasis occurs most frequently. Metastatic spread constitutes the primary source of morbidity and mortality, and the prognosis of patients with metastasizing lung cancer leaves much to be desired. Therefore, a thorough understanding of the organ-specific metastatic process, including lymph node metastasis and distant metastasis, is likely to be crucial to developing effective new therapies for lung cancer.While nowadays the reports on the role of the chemokine-receptor axis in organ-specific metastasis, especially brain-specific metastasis, of lung cancer were very rare. Then this study was to investigate the relationship between CCR7 expression and lymph node metastasis of NSCLC by RT-PCR, immunohistochemistry, and western blot assays and explore the mechanism of lymph node metastasis of lung cancer. And then this study was the first time to investigate the correlation between CXCR4 expression and brain-specific metastasis of NSCLC and explore the mechanism of brain metastatic specificity of lung cancer initially.Part I Effect of CCR7 and VEGF-C on lymph node metastatic potentiality of non-small cell lung cancerObjective:The incidence of lymph node metastasis of lung cancer was shown to be positive correlated with tumor chemotactic migration and lymphangiogenesis. This study was to investigate the relationship between lymph node metastatic potentiality of non-small cell lung cancer(NSCLC) and expression of CCR7 and VEGF-C.Methods:The samples of cancer tissues, lymph nodes and normal lung tissues from 65 patients with NSCLC, who underwent complete resection in Provincial Hospital Affiliated to Shandong University from January 2008 to August 2008, were collected. And all samples were examined by RT-PCR, immunohistochemistry and western blot assays to detect CCR7 and VEGF-C expression. Monoclonal antibody D2-40 was used to assessment of lymphatic vessel density (LVD). The SSPS 11.5 software package was used for statistical analysis. According to the clinicopathologic factors, the difference of CCR7 and VEGF-C expression was compared byχ2 test, and the difference of LVD was compared by u-test. Logistic regression analysis was performed to determine the independent risk factors of influencing lymph node metastatic potentiality of NSCLC.Results:CCR7 mRNA and VEGF-C mRNA expression was observed in 47(72.3%) and 39 (60.0%) lung cancer tissues by RT-PCR assay; CCR7 protein and VEGF-C protein expression was observed in 43 (66.2%) and 36 (55.4%) lung cancer tissues by immunohistochemistry; and CCR7 protein and VEGF-C protein expression was also observed in 44 (67.7%) and 37 (56.9%) lung cancer tissues by western blot, respectively. No significant difference was found between CCR7 and VEGF-C expression(P>0.05), and each was positive related to lymph node metastasis of NSCLC (P<0.05), furthermore co-expression of CCR7 and VEGF-C expression was more significantly related to lymph node metastasis of NSCLC (P<0.05). In lung cancer tissues, LVD was higher than that in normal lung tissues (P<0.001); and LVD in lung cancer was positively related to CCR7 expression, VEGF-C expression and lymph node metastasis. The expression rate of CCR7 mRNA and VEGF-C mRNA in metastatic lymph nodes were 85.7% and 74.3%, which were higher than that in non-metastatic lymph nodes(20.0% and 13.3%). Obvious expression of CCR7 and VEGF-C protein was found in no normal lung tissuse. Logistic regression analysis revealed that CCR7 mRNA expression (OR=10.275, P=0.005) and VEGF-C mRNA expression in tumors(OR=5.550, P=0.021) were independent risk factors of lymph node metastasis of NSCLC.Conclusions:CCR7 and VEGF-C were highly expressed in lung cancer and the metastatic lymph nodes, with significant correlation with lymph node metastasis of NSCLC. The co-expression of CCR7 and VEGF-C might promote lymphatic metastasis of lung cancer and might be a clinical predictor of lymph node metastasis of patients with NSCLC.Part II High-level CXCR4 expression correlates with brain metastasis of non-small cell lung cancerObjective:Lung cancer might develop organ-specific metastasis, and brain metastasis occurs most frequently, while the mechanism is still unclear. This study was to investigate the correlation between CXCR4 expression and brain metastasis of non-small cell lung cancer. Methods:The metastatic brain tumors and lung cancer tissues from 32 patients with solitary brain metastasis of NSCLC (M1), who underwent combined surgical treatment from June 1998 to September 2008, and 32 paring patients without distant metastasis (MO) and 30 patients with primary brain tumor, were examined by immunohistochemistry to detect the expression of CXCR4 protein. The difference of CXCR4 expression was compared byχ2 test. Estimation of survival was calculated using the Kaplan-Meier method, and the statistical differences were analyzed using the Log-rank test. Cox regression analysis was performed to identify risk prognostic factors of patients with solitary brain metastasis of NSCLC.Results:CXCR4 protein expression was observed in 29 (90.6%) lung cancer with brain metastasis(M1) and in all metastatic brain tumors (100%), and no difference in them two(P=0.238). And CXCR4 protein expression was observed in 68.8%(22/32) paired lung cancer without brain metastasis(MO), which was significantly lower than that in M1 lung cancer(P=0.000). In 63.3%(19/30) primary brain tumors, CXCR4 protein expression was observed and was significantly lower than that in metastatic brain tumors(P=0.000). The 1-year,3-year and 5-year cumulative survival rates of 32 patients with solitary brain metastasis of NSCLC were 62.5%,25%, and 15.6%; and the survival rates of patients with M0 NSCLC were 84.4%,53.1%, and 40.6%; Log-rank test showed that the rates of the two groups was significantly different(P=0.002). The 1-year,3-year and 5-year cumulative survival rates of 18 patients with synchronous brain metastasis were 72.2%,27.7%, and 16.7%; the survival rates of 14 patients with metachronous brain metastasis were 50%,21.4%, and 14.3%; Log-rank test showed there was no significant difference between the two groups(P=0.39). Cox regression analysis showed that gender, age, tumor histologic type or differentiation, T status, N status, brain metastasis pattern, adjuvant therapy and CXCR4 expression in lung cancer did not significantly affect survival of M1 NSCLC patients(P>0.05).Conclusion:CXCR4 protein expressed highly in lung cancer tissues and metastatic brain tumors of patients with M1 NSCLC, which indicated that CXCL12/CXCR4 signaling axis might play a chemotactic role in brain specific metastasis of NSCLC.