| BackgroundPancreatic ductal adenocarcinoma(PDAC)is one of the most lethal cancers.It is characterized by remarkably dense and firm desmoplasia.CAFs are the major contributor to desmoplasia,producing ECM and multiple soluble factors that lead to tumor progression.Recently,Studies with sc RNA-seq,genetically engineered mouse models have revealed that CAFs have heterogeneity in functional characterization,molecular expression,and spatial distribution.Therefore,gaining a comprehensive view of the heterogeneity of CAFs could identify new therapeutic vulnerabilities.CD144(CDH5,also known as vascular endothelial cadherin,VE-cadherin)is the major cadherin in endothelial cells but it is not expressed in the normal epithelium.Induction of CD144 in epithelial cancer cells may increase their intravasation as an early step in metastasis,and CD144 induction is responsible for vasculogenic mimicry in aggressive melanomas.Upregulation of CD144 was observed in invasive human breast tumors and in a breast cancer mouse model.In the heart and brain,fibroblasts can upregulate of CD144 expression under serum deprivation.However,whether CD144~+CAFs exist in hypoxic and ischemic TME in PDAC,and their effect on tumor cell remains unclear.Therefore,we designed and performed this study to explore the function and mechanism of CD144~+CAFs in PDAC.Methods1.CD144 IHC and mIHC was performed in the PDAC tissue sections to analyze the expression level of CD144 protein in CAFs;2.The clinical and pathological information of PDAC TMA were used to analyze the relationship between the expression of CD144 in PDAC stroma and OS/RFS,clinicopathological parameters.3.Outgrowth method was used to isolate the primary CAFs.CD144~+CAFs were sorted by immunomagnetic beads and confirmed by flow cytometry.4.Co-culture assay,transwell assay,wound-healing assay,EdU proliferation assay,western blot and mouse orthotopic transplantation tumor models were employed to explore the function of CD144~+CAFs.5.Western blot,gene knockdown with si RNA,blocking assay with specific small-molecule inhibitors and qPCR were conducted to illustrate the mechanism of crosstalk between CD144~+CAFs and cancer cells.Results1.The IHC and mIHC results suggested that there have been CD144~+CAFs in PDAC stroma.After analyzing the information of TMA,we have found that CD144 expression in PDAC stroma correlated with poor survival of PDAC patients.Flow cytometry analysis demonstrated that the percentage of CD144~+CAFs ranged from 8.32% to 68.4% of the stromal fibroblasts in 15 PDAC cases.2.We sorted CD144~+CAFs by immunomagnetic beads and confirmed by flow cytometry.We co-cultured pancreatic cancer cells with CD144~+CAFs.The proliferation,migration and invasion ability of tumor cells were dramatically enhanced upon co-cultured with CD144~+CAFs rather than CD144~-CAFs.3.To verify the above findings in vivo,we employed a xenograft mouse model by orthotopic co-injecting CAFs and pancreatic cancer cells into the pancreas of immunocompromised mice.Co-injection of BxPC-3 cells with CD144~+CAFs dramatically enhance the tumor growth.4.To understand how CD144~+CAFs exert their functions,we compared the cytokine profiles secreted by CD144~+CAFs and CD144~-CAFs using antibody microarrays and identified a panel of cytokines abundantly produced by the CD144~+CAFs.Among them,IL-6,VEGF,SDF-1α and Osteopontin have been reported to mediate pancreatic cancer proliferation,migration and invasion.5.To unravel the signaling pathways activated in CD144~+CAFs that sustained the production of IL-6,VEGF,SDF-1α and Osteopontin,we searched Pubmed and found that β-Catenin/STAT3 signaling pathway activation may result in upregulation of IL-6,VEGF,SDF-1α and Osteopontin.Therefore,we performed western blot and our data verified that β-Catenin/STAT3 signaling pathway was activated in CD144~+CAFs.Moreover,when we used a specific p-STAT3 inhibitor,the expression of CD144,IL-6,VEGF,SDF-1α and Osteopontin was downregulated accordingly.ConclusionWe demonstrated that CD144~+CAFs exist in PDAC stroma and they correlated with poor survival in cohorts of PDAC patients.CD144~+CAFs promoted tumor proliferation,migration and invasion by secreting IL-6,VEGF,SDF-1α and Osteopontin.Mechanistically,CD144~+CAFs are driven by persistent β-Catenin/STAT3 activation.Our study reveals a functional CAF subset that can be defined and isolated by specific cell-surface markers and suggests that targeting the CD144~+CAF subset could be an effective therapeutic strategy against PDAC. |
