Blockades Adenosine Receptor 2B Suppresses Pancreatic Adenocarcinoma Progression by Inhibiting Leukemia Inhibitory Factor Secretion from Macrophages

Background Pancreatic adenocarcinoma (PDAC) is a lethal disease with a five-year survival rate of less than 10%. The immunosuppressive tumor microenvironment (TME) was primarily responsible for the poor prognosis in PDAC. M2 Macrophages are a crucial cell population with pro-tumorigenic effects in response to extrinsic signals. Adenosine, a purine nucleoside catabolite of ATP, is one of the standard signals in TME that drives macrophage M2 polarization by activating adenosine receptor (ADOR). Although four types of ADOR have been reported previously, it is still unclear which receptor mediates the main pro-cancer effects in PDAC. Methods The conditioned medium (CM) was made by supernatants from ADOR-activation macrophages. The wound healing, trans-well, and CCK-8 assay detected the phenotypic change of pancreatic cancer cell lines PANC-1 and BxPC-3. The transcriptome sequencing was performed to screen the specific cytokine secreted from ADOR-activation macrophages. The ELISA assay was used to verify the cytokine concentration in the supernatants of ADOR-activation macrophages. The Western blot was performed to explore the expression level of proteins related to EMT, cell cycle, and cytokine. The bioinformatics analysis was utilized to find the signaling pathways modulating cytokine secretion. Immunohistochemistry (IHC) was used to calculate the IHC score of the ADOR correlated with the cytokine secretion. The Kaplan-Meier analysis was conducted to predict the prognosis of PDAC patients according to the IHC score of ADOR. The receptor antagonists were used in vivo experiments for mechanism validation. Results The CM promoted PANC-1 and BxPC-3 migration, invasion, and proliferation. Leukemia inhibitory factor (LIF) was the specific cytokine contained in the CM with cancer-promoting capacity based on the result of bioinformatics analysis. The activation of ADORA2B elevated the LIF concentration in the macrophage supernatants through the RAF-MEK-ERK signaling pathway. The expression ratio of ADORA2B ranks second among the four types of ADOR in PDAC. The IHC score of ADORA2B in PDAC significantly correlates with overall and disease-free survival in PDAC patients. LIF stimulated PANC-1 and BxPC-3 migration, invasion, and proliferation by connecting with the LIF receptor (LIFR) and activating the JAK-STAT signaling pathway. The ADORA2B and LIFR antagonists decreased the tumor size and number of hepatic metastatic lesions in the pancreatic orthotopic implantation model. Conclusion Activation of ADORA2B promotes LIF secretion from macrophages through the RAF-MEK-ERK signaling pathway. Meanwhile, the LIF secreted from macrophages promotes PDAC progression by activating the JAK-STAT signaling pathway.