The Mechanism Of EHF Deficiency Facilitates Gr-MDSC Infiltration To Promote Malignancy Of Pancreatic Ductal Adenocarcinoma

BackgroundESE3,also known as EHF,is a member 3 of the ETS homologous factor/epithelial specific ETS factor family.It is a member of the ETS superfamily on E26 and is mainly expressed in epithelial tissues.A large number of literatures have reported that EHF plays a key role in cell differentiation and tumor progression in different tumor types [1-2].Pancreatic ductal adenocarcinoma(PDAC),as one of the most malignant solid tumors,has extremely high invasiveness,and its most significant characteristics are early metastasis and chemotherapy resistance [3].The stroma of pancreatic ductal adenocarcinoma is particularly rich,in which a large number of astrocytes and immune cells are infiltrated,and most of the stromal cells are derived from bone marrow cells.As the main component of immunosuppressive cell population,MDSC,also known as bone marrow-derived immunosuppressive cells,is a heterogeneous population of immature bone marrow cells.It is generally believed that it mainly includes monocyte bone marrow-derived immunosuppressive cells(Mo-MDSC/M-MDSC),granulocytic bone marrow-derived immunosuppressive cells(Gr-MDSC/PMN-MDSC)and early immature bone marrow-derived immunosuppressive cells(e-MDSC)[4].These cells are mainly induced and differentiated in bone marrow,and will be attracted to the local microenvironment of tumor under the action of chemokines secreted by tumor cells and stromal cells,eventually leading to malignant phenotype of tumor,such as promoting epithelial mesenchymal transition,angiogenesis and tumor stemness [5-7].The role of MDSC in PDAC has been reported in recent years,such as holokai L’s studies have shown that PMN-MDSC infiltrating into tumor tissue can significantly inhibit the functional activity of T cells,thus providing a potential target for immunotherapy of pancreatic cancer [8] and our previous research team found that there were different degrees of MDSC population infiltration in pancreatic cancer when studying the effect of tumor suppressor gene EHF on the immune panorama of pancreatic cancer,and there was a significant negative correlation with tumor suppressor gene EHF [9].Gr-MDSC,a group of myeloid derived immunosuppressive cells with polymorphonuclear granulocyte,is the most common subgroup of MDSC.There are few reports on its association with pancreatic cancer,and the question whether EHF can affect the invasion and malignant promotion of this subgroup has not been reported.In the previous experimental analysis,we found that compared with normal pancreatic tissue and pancreatic ductal adenocarcinoma adjacent tissue,the proportion of Gr-MDSC infiltration in cancer tissue was significantly higher.Therefore,the objectives of this study includes the following aspects:(1)to clarify the correlation between EHF and the proportion of Gr-MDSC infiltrating in PDAC tissue(2)to explore the specific molecular mechanism of EHF affecting Gr-MDSCs’ infiltration(3)to verify the classical immunosuppressive function of this subgroup: whether it affects the proliferation and functional activity of CD8+T cells(4)to comprehensively explore the effect of Gr-MDSC on pancreatic cancer malignant reaction of tumor cells: will it significantly affect proliferation of tumor cells?Method:1.Prospective study: Fresh PDAC tissue samples were collected for flow cytometry detection of Gr-MDSC and immunohistochemical staining of EHF to analyze the correlation between the proportion of Gr-MDSC infiltration and the expression of EHF in pancreatic ductal adenocarcinoma.2.Retrospective study: 96 cases of tissue sections of PDAC patients from 2010 to 2020 were collected,EHF and CD15 immunohistochemical staining and immunofluorescence multi-label staining were performed,and then the correlation between EHF expression level and Gr-MDSC infiltration level was analyzed.3.Animal experiment: The stable PANC02 empty vector and EHF over expression/knock down cell lines were constructed and verified for subcutaneous tumorigenesis in C57/BL6 mice.Ratio of Gr-MDSC in tumor cell suspension of each group was detected to determine the effect of EHF expression level on Gr-MDSC infiltration level.4.Exploration of influence mechanism: The chemotactic co-culture of Gr-MDSC purified by magnetic beads and pancreatic cancer cells with different EHF expression was carried out.It was clear that EHF could affect the chemotaxis of Gr-MDSC and then lead to the difference of infiltration of this subgroup of cells in tumor tissues.5.The specific molecular mechanism of EHF affecting the chemotaxis of Gr-MDSC: WB and q PCR trials were used to detect the basic expression of EHF in the existing immortalized pancreatic cancer cell lines.On this basis,the cell lines stably over-expressing or knocking-down of EHF were constructed.The relationship between EHF and CXCL1 was verified by Western blotting/enzyme-linked immunosorbent assay(ELISA)/tissue immunohistochemistry and immunofluorescence.The direct negatively-transcriptional regulation of CXCL1 by EHF was further confirmed by CHIP experiment and double luciferase experiment.6.The density gradient separation solution(Ficoll)was used to separate and purify Gr-MDSC by magnetic beads,and the separation efficiency was tested.Then,Gr-MDSC with high purity(more than 90%)was used to co-culture with pancreatic cancer cells,and the classical proliferation function tests were used to detect whether it would produce malignant reaction on tumor cells,and then it was co-cultured with CD8+T cells to explore the classical immunosuppressive function of this subgroup of cells in pancreatic cancer.Results:1.20 cases of fresh PDAC specimens and 96 cases of retrospective tissue sections were prospectively studied.By immunohistochemical staining of EHF and flow cytometry to mark the proportion of infiltration of Gr-MDSC(CD45+/HLA-DRlow/-/CD33+/CD11b+/CD15+Gr-MDSC),it was found that there was a significant negative correlation between the expression of EHF in tumor cells and the proportion of Gr-MDSC infiltration in corresponding tissues.2.The correlation between the expression of EHF and the number of Gr-MDSC infiltration in tumor tissue was determined by animal experiments in vivo.The subcutaneous tumor formation model was established in C57/BL6 mice using PANC02 empty vector and EHF overexpression and knock-down stable cell lines.The results showed that the proportion of Gr-MDSC infiltrating in tumor tissue of EHF overexpression group was significantly reduced.3.In vitro chemotaxis experiment confirmed that the loss of EHF could cause the chemotaxis of Gr-MDSC to increase significantly.4.The regulatory mechanism between EHF and CXCL1 was confirmed.Firstly,at the cellular level,Western blotting,ELISA and PCR experiments confirmed that there was a significant negative correlation,that is,the expression of CXCL1 was significantly increased in the EHF down-expression group;secondly,at the tissue level,western blotting was performed on the cancer and adjacent tissues.The results showed that there was a significant co-localization between EHF and CXCL1 at the tissue level,and there was a significant negative correlation.Finally,it was confirmed that EHF could bind to the promoter region of CXCL1 and negatively-regulate the expression of CXCL1 by chromatin immunoprecipitation experiment and double luciferase experiment.5.Co-culture of Gr-MDSC with CD8+T cells to verify its immunosuppressive function: the proliferation of CD8+T cells was significantly inhibited,and the apoptosis of CD8+T cells was significantly promoted,and the secretion of IFN-γ was inhibited.6.Co-culture of Gr-MDSC with tumor cells to determine its mechanism of promoting tumor malignancy.The results showed that the proportion of EDU and Ki67 positive cells was significantly increased,and the expression of PCNA and Ki67 was significantly increased.Conclusion:1.There is a significant negative correlation between the expression of EHF and the infiltration proportion of Gr-MDSC in PDAC tissue.2.EHF can directly negatively-regulate the expression of CXCL1.3.EHF can affect the chemotaxis and recruitment of Gr-MDSC by regulating the expression of CXCL1.4.Infiltrating Gr-MDSCs in PDAC has classical immunosuppressive function.5.Infiltrating Gr-MDSC in PDAC can significantly promote the proliferation of pancreatic cancer cells.