The Impact Of Atypical Chemokine Receptor CCRL1on Biological Behaviors Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most common malignant tumors[1]. There is a high prevalence of HCC in China, accounting for more than a half of the patients in the world[2]. Although much progress has been made in HCC research, the overall curative effect is not satisfied due to the rapid progression, high recurrence and metastasis rates. Untill now, tumor resection and liver transplantation are the most effective and standard therapies for HCC[3]. The5-year recurrence rate after surgery is up to50%to70%. Therefore, it is essential to explore the underlying mechanisms of recurrence and metastasis of HCC to get a better understanding about tumor invasion and metastasis, and to finding effective prophylaxes and therapies against tumor.Tumorigenesis, development, invasion and metastasis of tumors are complex and dynamic processes. Only combining tumor with microenvironment can we have a deep insight of the the mechanisms which involve the collaboration of chemokines, growth factors, cell adhesion molecules, extracellular proteases and angiogenesis factors. Chemokines and their receptors have a close relationship with tumor growth, invasion, angiogenesis, and metastasis.In this study, we discussed the impact of atypical chemokine receptor CCRL1on biological behaviors of HCC. Tissue microarray for240HCC patients showed decreased expression of CCRL1in tumor cells. Moreover, the patient with low expression of CCRL1have a poor survial than the higher one. We use lentivirus transfection technique to regulate the expression of CCRL1in HCC cell line. By in vivo and vitro experiment, we observed the impact of CCRL1expression on tumor growth, proliferation, invasion and metastasis. At last, we discussed the probable mechanisms for the anti-tumor effects of CCRL1, providing new target for HCC prevention and treatment.Part Ⅰ The expression pattern of CCRL1and the clinical significances in HCCIn this part, we focused on the expression of CCRL1in tumor and peri-tumor tissues and the relations between CCRL1expression and patient prognosis.Firstly, immunohistochemical staining was performed in tumor and peri-tumor tissue of240patients who underwent HCC resection. Image-Pro Plus6.0was used to calculate the mean optical density of the immunohistochemical staining. After that, we detected the expression of CCRL1in six HCC cell lines by western blot and real-time RT-PCR. At last, the clinical data we analysed with the expression of CCRL1in order to explore the value of CCRL1as a prognostic factor for HCC.We found that the expression of CCRL1was decreased in tumor compared to peri-tumor (P=0.015), which indicated a relative deletion of CCRL1in tumor tissue. The various expression levels of CCRL1were observed HCCs. Especially for MHCC97H(97H), MHCC97L(97L) and MHCCM3(M3), on the rise of metastatic potentials of these three cell lines(97L<97H<M3), the expression of CCRL1decreased gradually (97L>97H>M3), which implied that CCRL1might have the capability to inhibit tumor invasion and metastasis. Together with the follow-up data of the240patients, we found that the low expression of CCRL1was corelated with vascular invasion (P=0.038) and poor differentiation (P=0.037). The patient with low CCRL1expression had a low overall (P＜0.001) and recurence-free (P=0.002) survival rate than high expression group. Multi Cox regression showed that CCRL1together with tumor number and vascular invasion was an independent predictor for overall (hazard ratio, HR=0.579;95%confidence interval, CI=0.411-0.816;P=0.002) and recurrence-free survival(HR=0.657;95%CI=0.462-0.934;P=0.019) rates.Part Ⅱ The impact on HCC biological characteristics by regulation of CCRL1expressionIn order to explore the impact of CCRL1on tumor growth, migration, invasion and metastasis in vivo and in vitro experiments.Using lentivirus vector, we transfected shCCRL1knock-down and over-expression plasmids in97L and M3cell lines, respectively, to constructed stable cell lines of97L-shCCRLland M3-CCRL1. The proliferation was measured by Cell-IQ system, migration ability by scratch test, invasion ability by transwell assay. We next explored the carcinogenesis and pulmonay metastasis abilities of the cells using subcutaneous and in situ xenotrans models in nude mice. In addition, we tested the quantity of CCL19and CCL21in the supernatant of cells and in situ tumor by ELISA.In vitro, our results showed that, down-regulation of CCRL1caused the intensive proliferation, invasion, and migration of HCC cells as well as the quantity of CCL19and CCL21. On the contrary, over-expression of CCRL1inhibited the proliferation, invasion and migration of HCC cells, reduced the quantity of CCL19and CCL21. In vivo, the volume of the xenotrans in the low expression group surpassed that in the high expression goup and more pulmonary metastases were detected in low expression group.CCRL1can inhibit the proliferation, invasion and migration of the HCC cell lins, indicating that CCRL1might become a new target for the treatment of HCC.Part III The underlying mechanisms of CCRL1’s impact on HCCIn order to understand how CCRL1inhibited the proliferation, invasion and migration of HCC, we discussed the underlying mechanism in two aspects from tumor itself and microenvironment. On one hand, CCRL1scavenged its ligands (CCL19,CCL21) to affected the function of other receptors (CCR7) for tumor killing. On the other hand, the over-expression of CCRL1can up-regulate some chemokines to recruit lymphocytes. Here, we mainly focused on the B lymphocytes that infiltrate into the tumor.We first performed immunohistochemical staining on CCRL1, CCL19, CCL21and CCR7. Also, cell signaling pathways of the97L-shCCRL1was detect by western blot. Using siRNA to down-regulate CCR7, we detected the change of proliferation and invasion in HCC cells. Furthurmore, we seperated CD20+B lymphocytes by discontinuous gradient centrifugation, and tested the phenotype and cytokines by flow cytometry. We co-cultured the tumor cells with CD20+B lymphocytes to explore its direct tumor-killing effect.IHC showed that the expression of CCRL1had a negative correlation with that of CCL19, CCL21and CCR7. The results of western blot indicated that the phosphorylation of Akt/GSK-3β increased significantly by down-regulating CCRL1, which indicated that CCRL1might regulate tumor proliferation and invasion through this signaling pathways which could be blocked siCCR7and neutralizing antibodies of CCL19/21. The majority of tissue infiltrating B cells were enriched in margin area and the B cells there had a IgD-IgG+CD27-CD38-phenotype. They produced INF-γ and IL-12, and they could directly kill the tumor cells by granzyme B and TRAIL. The density of marginal B cells was correlated with the prognosis of HCC patients.In summary, up-regulating CCRL1of tumor cells decreased the quantity of CCL19and CCL2to inhibit CCR7, and then reduced the phosphorylationthe of Akt/GSK-3β.In additon, up-regulating CCRL1of tumor cells recruited CD20+B lymphocytes to exert tumor-killing effect.