Pancreatic Cancer-derived Exosomes Promote Liver Pre-metastatic Niche Formation Via Mertk^hiKupffer Cell Subset

Pancreatic cancer is one of the most aggressive malignant tumors,and the liver is the organ most prone to metastasis.Patients with liver metastases from pancreatic cancer have a worse prognosis than patients with localized tumors,and they are often not recommended for surgical resection of the primary tumor.Kupffer cells are liver-resident macrophages located in the hepatic sinusoids and account for approximately 10%of the total number of cells in the liver.In the early stages of tumor metastasis,Kupffer cells are able to remove cancer cells through phagocytosis,thus protecting the liver from metastatic tumors.However,Kupffer cells can also promote tumor cell proliferation and angiogenesis by secreting substances such as human hepatocyte growth factor（HGF）and vascular endothelial growth factor（VEGF）.In addition,Kupffer cells recruit other cells that promote tumor metastasis,such as neutrophils,monocytes,or myeloid-derived suppressor cells（MDSCs）,to induce an immune tolerance microenvironment in the liver.In 2015,Costa Silver et al.showed that Kupffer cells take up exosomes from pancreatic cancer,which caused hepatic stellate cells to secrete transforming growth factor-β（TGF-β）and express fibronectin.The deposition of fibronectin led to the formation of a hepatic fibrotic microenvironment,which promoted the recruitment of myeloid-derived cells,creating a microenvironment conducive to tumor cell growth and immune escape,and promoting tumor metastasis.Based on the above research background,we constructed a mouse model of pancreatic cancer liver metastasis.We isolated CD45-positive non-parenchymal cells from mouse liver tissue and performed single-cell sequencing.As a result,a subset of Kupffer cells with high expression of myeloid-epithelial-reproductive tyrosine kinase（Mertk）was found in liver metastases from pancreatic cancer.We constructed an exosome-educated mouse model and found that pancreatic cancer-derived exosomes can activate the mitogen-activated protein kinase（MAPK）signaling pathway,which induces the production of Mertk^hiKupffer cells.Mertk^hiKupffer cells release multiple chemokines which can recruit MDSCs,thus creating an immunosuppressive microenvironment conducive to tumor cell growth.In this study,we found and identified the Mertk^hiKupffer cell subset which can be induced by pancreatic cancer-derived exosomes.We also explored the function and mechanism of this subset in the formation of the pre-metastatic niche in the liver.The present study is expected to provide new ideas on the mechanism of development and early intervention of liver metastases from pancreatic cancer.PartⅠDiscovery and identification of Mertk^hiKupffer cell subset in liver metastases from pancreatic cancerObjective:To investigate the phenotypic changes of Kupffer cells during liver metastasis of pancreatic cancer.Methods:1.The presence and distribution of Kupffer cells in liver metastases of pancreatic cancer were clarified by immunohistochemistry and immunofluorescence staining.2.We constructed a mouse model of pancreatic cancer liver metastasis by spleen injection of tumor cells.The liver metastasis of the model was analyzed by animal imaging and morphological observation.3.We sorted CD45-positive cells in mouse liver tissue by magnetic beads and then performed single-cell sequencing.4.We developed and validated a flow-circle gating strategy for mouse Kupffer cells.Detection of Mertk expression in mouse Kupffer cells by using flow cytometry.Validation in human pancreatic cancer liver metastasis tissue by using immunofluorescence staining.Results:1.CD68-positive Kupffer cells were significantly clustered around human pancreatic cancer liver metastases compared to CD4,CD8 and CD20-positive immune cells.2.More CD68-positive Kupffer cells clustered around human pancreatic cancer liver metastases compared to the adjacent tissue.3.Three weeks after spleen injection of tumor cells,animal imaging showed that mice developed liver metastases.Morphological observation showed tumor metastases on the liver of mice.4.Single-cell sequencing results showed a subset of Mertk^hiKupffer cells in liver metastases from pancreatic cancer in mice.5.Flow analysis showed that elevated Mertk expression in kupffer cells in mouse pancreatic cancer liver metastasis tissue.Immunofluorescence results showed the presence of Mertk^hiKupffer cell subset in human pancreatic cancer liver metastasis tissue.Conclusion:Pancreatic cancer can recruit CD68-positive Kupffer cells during liver metastasis.It can also induce the formation of Mertk^hiKupffer cells.PartⅡPancreatic cancer-derived exosomes induce the production of Mertk^hiKupffer cell subsetObjective:To investigate the effects of pancreatic cancer-derived exosomes on Kupffer cells and the pre-metastatic niche of the liver.Methods:1.We extracted pancreatic cancer-derived exosomes by ultracentrifugation,and then examined the exosome morphology and particle size by electron microscopy and NTA.2.We constructed an exosome-educated mouse model by tail vein injection.We used membrane dyes to label exosomes and detected exosome enrichment by in vivo imaging of small animals.Expression of pre-metastatic gene was detected by real-time fluorescence quantitative PCR and immunohistochemistry.3.We extracted Kupffer cells by liver perfusion and density gradient centrifugation.The morphology of Kupffer cells was observed under microscope and the purity of Kupffer cells was detected by flow cytometry.4.Kupffer cells were stimulated by exosomes in vitro,and the expression level of Mertk was detected by flow cytometry.5.Flow cytometry,western blot and immunohistochemistry were used to detect the expression of Mertk in liver tissues of exosome-educated mice.The expression of Mertk in mouse hepatocytes and Kupffer cells was detected by real-time fluorescence quantitative PCR.The expression and co-location of F4/80 and Mertk in mouse liver tissues were detected by immunofluorescence.6.We searched the activation pathway of Mertk^hiKupffer cells by KEGG enrichment,and then verified it by western blot.Results:1.The results of electron microscopy and NTA showed that the extracted disc-shaped vesicle bodies with a diameter of 30-150nm could be identified as exosomes.2.The results of small animal imaging showed that liver was the main organ of exosome enrichment in mice.3.After educated by cancer-derived exosomes,the gene expression level of pre-metastatic niche S100a8,S100a9,Bv8,Mmp9 and Fibronectin increased.4.The purity of Kupffer cells isolated from mouse liver was over 90%,and they had typical morphological characteristics.5.Pancreatic cancer-derived exosomes can induce the expression of Mertk in mouse primary Kupffer cells.6.Mertk^hiKupffer cells were found in liver tissues of exosome-educated mice.7.KEGG enrichment analysis revealed that MAPK pathway was activated in Mertk^hiKupffer cells,and exosomes could induce phosphorylation of ERK,JNK and P38proteins in Kupffer cellsConclusion:Pancreatic cancer-derived exosomes can activate the MAPK signaling pathway of Kupffer cells and induce the production of Mertk^hiKupffer cells.PartⅢThe roles and mechanisms of Mertk^hiKupffer cell subset in the pre-metastatic niche of the liverObjective:To investigate the function of Mertk^hiKupffer cell subset.Methods:1.The expression of chemokines in Kupffer cell subsets was analyzed by single cell sequencing data,and verified by real-time quantitative PCR.2.Flow cytometry and immunofluorescence were used to detect the effect of exosomes on the level of MDSCs in mouse liver.We detected the distribution of Mertk^hiKupffer cells and MDSCs in hepatic metastasis specimens of human pancreatic cancer by immunofluorescence and immunohistochemistry.3.MDSCs and Kupffer cell subsets were sorted by flow cytometry,and then co-cultured through transwell.The recruitment effect of different Kupffer cell subsets on MDSCs was detected by flow cytometry.Results:1.Mertk^hiKupffer cell subset up-regulated Ccl2,Ccl3,Ccl4 and other chemokines.2.Exosome-educated can promote the recruitment of MDSCs in mouse liver.3.The proportion of Mertk^hiKupffer cell subset and MDSCs around human liver metastasis was significantly higher than that in adjacent tissues.4.Compared with Mertk^loKupffer cell subset,flow cytometry results showed that Mertk^hiKupffer cell subset could recruit MDSCs significantly.Conclusion:Mertk^hiKupffer cell subset can promote the recruitment of MDSCs in the liver by releasing chemokines,thus inducing the formation of pre-metastatic niche of the liver.