Relationship Between CCL20/CCR6/TH17Axis And Vascularinvasion And Metastasis In Patients With Primary Hepatocellular Carcinoma

Background and ObjectiveHepatocellular carcinoma(HCC) is one of the common types of malignancies inChina. Lack of effective treatment options,surgical resection is still the mainapproaches in clinicalso far. Because of intrahepatic metastasis,mainly vascularmetastasis, the rate of recurrence or metastasis after surgery is extremely high and the5-year survival of HCC is very low. Our recent work found that except tumor tissueitself,mainly tumor size, influenced recurrence of HCC patients after surgery,elevated pretherapy serum IL-17correlated to increased risk of early recurrence aftercurative hepatectomy, and IL-17is mainly produced by tumor-infiltrating Th17cells.Several studies including in China have documented that IL-17were able to promoteangiogenesis and tumor growth, and the human Th17cells in tumor micro-environments were expanded. The mechanisms of Th17cell recruitment in tumormicroenvironment is still unclear and their roles on tumor vascular invasion in clinicalneed to be investigated.Chemokines are small molecular peptides with molecular weight of8-14KD.Theyare master regulators of immune cell trafficking in the body.Chemokines interactwith seven trans-membrane-G-protein-coupled receptors to exert their chemotactic onimmune cell in specific and non-specific immune response. The interaction ofchemokines and their receptors are involved in a variety of biological functions inphysiological and pathological conditions. Th17cells express chemokine receptorCCR6and exclusively responds to chemokine CCL20. In this study, we investigatedthe relationship between CCL20/CCR6/Th17axis and HCC vascular metastasis.Methods: Expression levels of CCL20mRNA in cell lines of L-02, Hep3B, Huh7and HepG2were quantified by using SYBR Green real time PCR; CCL20secretionsfrom these cell lines were quantified by using ELISA. The chemotactic effect of HCC cell line Hep3B on human peripheral blood mononuclear cells was determined byusing transwell chemotaxis assay. Pretherapy serum levels of IL-1α，IL-1β，IL-6，IL-8，IL-10，IL-17，IL-23，IFN-γ, TNF-αand CCL20in93patients with HCC weremeasuredby using9-plex array and ELISA. All the patients were chronic hepatitis Bvirus associated HCC and51cases were those with vascular invasion and metastasis(metastasis group) and42cases were not (non-metastasis group). CCL20and CCR6mRNA expressions in HCC paired tumor and tumor-adjacent tissues were determinedby using SYBR Green real time PCR in41patients, among them,20ones were fromthe group of patients with metastasis and21ones were the group of patients withnon-metastasis. The CCL20expressions were further determined byimmunohistochemistry staining.Results: HCC cell lines expressed and secreted higher amount of CCL20, whicheffectively recruited CCR6+T cells. Pretherapy serum levels of CCL20increasedsignificantly in HCC patients than those with benign hepatic hemangiomas. Inaddition, CCL20and IL-17were significantly higher in HCC patients with metastasis(P<0.05) than those with non-metastasis. Serum levels of CCL20were positivelycorrelated with tumor sizes and serum levels of IL-17.The mRNA expression levels ofCCL20increased both in HCC tumors and tumor-adjacent tissues compared with thedonor’s normal liver tissues (P<0.001). However, the levels in the tumor tissues wassignificantly higher than that in tumor-adjacent tissues (P<0.01). CCL20mRNAshowed no statistically differences between the tumor tissues with metastasis andnon-metastasis. But the patients with metastasis showed significantly higherexpressions of CCR6both in their tumor and tumor-adjacent tissues than those withnon-metastasis.Conclusion:CCL20/CCR6/Th17axis may promote vascular invasionandmetastasis of HCC.