The Mechanism Underlying ESE3 Deficiency Mediated Tumor Mo-MDSC Accumulation To Enhance Malignancy Of Pancreatic Cancer

BackgroundESE3?ETS homologous factor/epithelium-specific ETS factor family member 3,EHF?is a member of the E26 transformation-specific?ETS?superfamily,mainly expressed in epithelial tissue and plays an important role in cell differentiation and tumor progression^[1,2].Pancreatic ductal adenocarcinoma?PDAC?is a highly aggressive malignancy characterized by early metastasis and chemotherapy resistance^[3].PDAC has a dense matrix of white blood cells,mainly bone marrow cells.These myeloid cells are nonhomogeneous and were mainly composed of mononuclear and granular^[4].MDSCs are produced in the bone marrow and recruited through the tumour-derived chemokines to the tumor microenvironment and promote tumor growth^[5-7].Recently,several reports have verified that mo-MDSC plays a role in a variety of tumors,but no one has yet reported its role in pancreatic cancer and the effect of ESE3 on mo-MDSC.In the previous work,we found that compared with normal pancreatic tissue,the infiltration percentage of mo-MDSC was significantly increased in PDAC cancer tissues;meanwhile,mo-MDSC can be counterproductive to tumor cells.Therefore,the purpose of our experiment is?1?to explore the relationship between ESE3 expression and mo-MDSC and to clarify its regulatory mechanism.?2?further to explore the effect of mo-MDSC on pancreatic cancer cells.Thus,the regulation of ESE3 on mo-MDSC and its effect on pancreatic cancer were determined from the level of clinical data,cell level and animal level.Method1.Mo-MDSC Flow cytometry detection and immunohistochemical staining of ESE3/CXCL1 were performed for pancreatic ductal adenocarcinoma specimens,and the correlation between ESE3 expression level and mo-MDSC infiltration ratio in pancreatic ductal adenocarcinoma was analyzed.2.Animal experiments PANC02 empty-vector and PANC02 with ESE3over-expression cell lines were built,and C57/BL mice were subcutaneous with them.At the same time,we set up a drug group,namely:Anti-Gr1 for the removal of mo-MDSC.After two weeks,and then collect tumors,collect single cell suspension,and streaming antibody staining,flow detection the change of mo-MDSC percentage.3.The basic expression level of ESE3 in pancreatic cancer cell lines was detected by Western blotting,and the stably-transfected cell lines of ESE3 were constructed.The relationship between ESE3 and IL-6/CXCL1 in protein level was tested by Western blottng.The changes of EMT and stemness related molecules in tumor cells were determined by WB.4.The CD14⁺monocytes were co-cultured with tumor cells.After 5 days,CD14/CD33/CD11b/HLA-DR staining were used in the co-cultured CD14⁺cells,and the relationship between the proportion of mo-MDSC and ESE3 was detected.Using Transwell system simulation process of CD14⁺monocyte chemotactic,after 90minutes,three-step later under a microscope and count,finally statistical analysis of the relationship between chemotaxis of CD14⁺cells and ESE3'effect in the chemotaxis.In this way,after 5-day'co-culturation we see how mo-MDSC impacts pancreatic cancer cells.5.The influence of ESE3 on the secretion level of CXCL1 was verified by ELISA,and the relationship between them was verified by RT-PCR at mRNA level.6.The regulational mechanism of ESE3 to CXCL1 was verified by CHIP.Results1.The relationship between ESE3 and mo-MDSC.There was a significantly negative correlation between ESE3 expression level and mo-MDSC infiltration ratio,and it was statistically significant.2.The relationship between ESE3 and mo-MDSC was verified by animal experiments.The proportion of mo-MDSC infiltrating in mouse tumor tissues of ESE3-overexpression group was significantly lower than that in the control group.At the same time,after the removal of mo-MDSC,the stemness ratio was generally decreased,but there was no significant difference between groups,and there was no statistical significance.3.In vitro co-culturation combined flow cytometry was used to verify the mechanism how ESE3 affects the change of mo-MDSC.We found that pancreatic cancer cells induces CD14?+?monocyte chemotaxis,and compared with the control,the number of chemotactic was obviously decreased in ESE3-overexpression group.Moreover,ESE3 also affects the induction of CD14⁺monocytes to mo-MDSC,and the induction ratio of ESE3 high expression group is decreased compared with the control group.More importantly,we found that mo-MDSC enhances the stemness and EMT of pancreatic cancer cells.4.The relationship between ESE3 and IL6/CXCL1 is determined by Western blotting and ELISA,they were significantly negatively correlated at protein levels.5.CHIP assay confirmed that ESE3 can directly and negatively regulates CXCL1.Conclusion1.The expression level of ESE3 was significantly negatively correlated with the infiltration number of mo-MDSC in pancreatic cancer tissues.2.ESE3 can directly and negatively regulates CXCL1.3.ESE3 can negatively regulates the chemotaxis of CD14⁺monocytes and the induction of mo-MDSC separately through CXCL1 and IL6.4.Mo-MDSC can enhance the EMT and stemness of tumor cells,and ESE3 high expression will reverse this effect.