CX3CL1 recruits CX3CR1pos macrophages to promote tumor progression in intrahepatic cholangiocarcinoma

Abstract

Background & aims Intrahepatic cholangiocarcinoma (iCCA) is the second most prevalent liver malignancy with a worse prognosis. It is characterized by a highly desmoplastic tumor microenvironment (TME) featured with abundant macrophages. Chemokines orchestrate the recruitment and infiltration of macrophages into the TME. The significance of CX3CR1pos macrophages with pro-tumoral features has been documented in several tumors other than iCCA. We aimed to investigate the role of the CX3CL1/CX3CR1 signaling in the progression of iCCA. Methodology CX3CL1 expression was analyzed in two independent clinical cohorts. The clinical significances of CX3CL1 and CX3CR1pos macrophages were evaluated by Cox proportional hazards model. Phenotypes and functions of tumor-infiltrated and monocyte-derived CX3CR1pos and CX3CR1neg macrophages were also assessed. The interactions between iCCA cells and CX3CR1pos macrophages were evaluated both in vitro and in vivo. The mechanisms for CX3CR1pos macrophages contribute to the immunosuppressive microenvironment and tumor invasiveness were investigated in cells and an orthotopic model. The therapeutic potential of a CX3CR1 antagonist (AZD8797) targeting the CX3CL1-CX3CR1 axis was explored in vivo. Results CX3CL1 was the most frequently up-regulated chemokine of iCCA in HKU and TCGA cohorts. Overexpression of CX3CL1 in iCCA tissue indicated tumor invasiveness and poor prognosis in both HKU and external validation cohorts. CX3CR1pos macrophages were independent risk factors for both disease-free and overall survival of iCCA. CX3CL1 facilitated the infiltration of CX3CR1pos macrophages into the TME of iCCA. CX3CR1pos macrophages not only contributed to immunosuppressive TME through secreting IL10, but also promoted an invasive phenotype of iCCA cells via CCL5-mediated epithelial-mesenchymal transition. It is demonstrated that the tumor volume of CX3CL1-high-expressing xenografts was markedly more extensive than those of CX3CL1-low-expressing xenografts. Higher rate of lung metastases was shown in the group with CX3CL1-high-expressing xenografts compared to the control group. The protumoral effects of CX3CL1 cannot be achieved without the presence of CX3CR1pos macrophages. Notably, blockage of the CX3CL1/CX3CR1 axis using AZD8797 inhibited the growth and metastasis of iCCA in vivo through the attenuation of immunosuppressive environment. Conclusions CX3CL1 promoted the recruitment of CX3CR1pos macrophages to the TME of iCCA, and this subpopulation of macrophages further facilitated iCCA progression by the induction of immunosuppressive environment. The identification of the CX3CR1pos macrophage subset may contribute to a better insight into the TME and provide a potential immunotherapeutic target for iCCA treatment.