Loss Of KDM6A Recruits Tumor-associated Neutrophils And Promotes Neutrophil Extracellular Traps Formation Via CXCL1-CXCR2 Axis In Pancreatic Cancer

Objective:Pancreatic cancer is one of the most malignant tumors,characterized by the immunosuppressive tumor microenvironment and dense desmoplastic stroma.KDM6A（Lysine（K）-specific demethylase 6A）is a frequently mutated tumor suppressor gene in pancreatic cancer,whose deletion promotes the growth and metastasis of pancreatic cancer.However,how KDM6A loss impacts the pancreatic tumor immune microenvironment is not known.This study aims to explore the effect of KDM6A deficiency on the tumor microenvironment of pancreatic cancer,elucidate its molecular mechanism,and provide potential therapeutic targets for KDM6A-deficient pancreatic cancer.Methods:1.Establish pancreas-specific Kdm6a-knockout Ptf1a^Cre;LSL-Kras^G12D/wt;LSL-p53^R172H/wt（KPC）genetically engineered mouse model.Detect different immune cells by immunohistochemistry and immunofluorescence.2.Establish KDM6A knockout pancreatic cancer cell line PANC-1 by using CRISPR/Cas9 system.Establish KDM6A knockdown pancreatic cancer cell line HPAF-Ⅱand human pancreatic normal ductal epithelial cell line HPDE by using si RNA.Establish mouse KDM6A knockdown pancreatic cancer cell line KPC 7940 by using sh RNA.Detect chemokine gene expression by RNA sequence in PANC-1 cells.Confirm chemokine m RNA and protein levels by RT-PCR and ELISA in all cell lines.Use immunohistochemistry to confirm chemokine levels in KPC mouse pancreatic cancer samples.3.Use the conditioned medium from KDM6A knockout or knockdown pancreatic cancer cells and corresponding immune cells to do chemotaxis assay in vitro to verify the recruitment of immune cells by KDM6A deficient pancreatic cancer cells.Verify the molecular mechanism by using the corresponding chemokine neutralizing antibody.4.Establish mouse pancreatic cancer orthotopic xenograft model and treat them with corresponding chemokine neutralizing antibody.Compare the tumor growth and detect immune cell infiltration by immunohistochemistry and immunofluorescence.Results:1.In the KPC mouse model,loss of KDM6A promoted the growth of pancreatic cancer.Furthermore,loss of KDM6A was associated with increased tumor-associated neutrophil（TAN）infiltration and neutrophil extracellular traps（NETs）formation in pancreatic cancer.2.In KDM6A knockout human pancreatic cancer cells PANC-1,the m RNA and protein levels of cytokines related to neutrophil chemotaxis such as CXCL1 and CXCL2 were increased,and the increase of CXCL1was the most significant.The m RNA and protein levels of CXCL1 were significantly increased in KDM6A knockdown human pancreatic cancer cells HPAF-II,human pancreatic normal ductal epithelial cells HPDE,and mouse pancreatic cancer cells KPC 7940.In the KPC mouse model,KDM6A deficient tumors showed significantly increased CXCL1expression,and tumor-infiltrating neutrophils expressed the CXCL1receptor,CXCR2.3.Human acute myeloid leukemia cells PLB-985 were differentiated into neutrophil-like cells by using DMSO to mimic the chemotaxis of neutrophils in vitro.The chemotaxis of neutrophil-like cells PLB-985 was significantly increased when using the conditioned medium from KDM6A knockout and knockdown human pancreatic cancer cells in vitro.The conditioned medium from KDM6A knockdown mouse pancreatic cancer cells KPC 7940 could significantly increase the chemotaxis of mouse bone marrow-derived neutrophils in vitro,while anti-CXCL1 neutralizing antibody could significantly reverse this effect.4.In the mouse pancreatic cancer orthotopic xenograft model,KDM6A deficiency promoted pancreatic cancer growth,along with increased neutrophil infiltration and NETs formation in cancer lesions.While the use of anti-CXCL1 neutralizing antibody reduced neutrophil infiltration and NETs formation in pancreatic cancer,and inhibited the growth of KDM6A-deficient pancreatic cancer.Conclusions:Loss of KDM6A in pancreatic cancer cells increased neutrophil infiltration and NETs formation in tumors by upregulating neutrophil-related chemokine CXCL1.When the CXCL1-CXCR2 axis was blocked by using anti-CXCL1 neutralizing antibody,neutrophil infiltration and NETs formation were reduced,and the growth of KDM6A deficient pancreatic cancer was inhibited.Therefore,the CXCL1-CXCR2 axis may become a potential therapeutic target for KDM6A deficient pancreatic cancer.