The Study Of Lipid Microenvironment Induces Macrophages Pyroptosis And Promotes Migration And Invasion Of Pancreatic Cancer Through NLRP3/IL-1β Axis

Pancreatic cancer(PC)is one of the most malignant tumors with poor prognosis.Lipid microenvironment has been confirmed as the independent risk factor for occurrence and development of pancreatic cancer.However,there are few studies focusing on relationship between lipid microenvironment and occurrence,evolution and prognosis of PC,and the mechanism is unclear.Macrophages in lipid microenvironment are the core of tumor immune metabolism,and macrophage-related chronic inflammatory response plays an important role in inducing formation of tumor microenvironment(TME).It is conducive to find novel diagnostic markers and therapeutic targets through exploring the effects of macrophages on the occurrence and development of PC,which has great significance to improve the early diagnosis rate and prognosis of PC patients.The study focuses on flowing two parts to explore the association between high lipid environment and pancreatic cancer:Part 1 The relationship of clinicopathological feature and prognosis between hyperlipidaemia and pancreatic cancerObjective:To explore the association between hyperlipidaemia and clinicopathological characteristics and the influence on prognosis of PC patients.Methods:1.The clinical and pathological data of 265 PC patients who received radical surgical treatment at Department of General Surgery,Qilu Hospital,Shandong University from January 2013 to September 2020 were collected and analyzed retrospectively.And follow-up information gained by telephone.2.Pearson χ2,Student’s t tests,variance analysis or Logistic regression was used to analyze the correlation between hyperlipemia and clinicopathological characteristics of pancreatic cancer,respectively.Kaplan-Meier survival curve was used to assessed the influence of hyperlipemia on prognosis of PC patients.Results:1.There were 115 PC patients was hyperlipemia accounting for(43.4%),and the most common form was increase of triglyceride(TG).2.In hyperlipemia group,PC patients with body mass index≥25 kg/m2 had higher proportion than normal lipid group(36.1%vs 21.2%,P=0.031);The proportion of carcinoma located at head of pancreas(83.5%vs 40.7%,P<0.001),staging of T1/T2(79.1%vs 60.7%,P<0.01)and lymphatic metastasis(36.5%vs 22.7%,P<0.01)were higher.3.In patients of PC,hyperlipemia were closely associated with tumor location(OR=10.529,P<0.01)and body mass index(OR=3.671,P=0.008).Subgroup analysis results showed that triglyceride,total cholesterol and high-density lipoprotein disorders were associated with tumor location(P<0.05).Meanwhile,TG disorder had association with body mass index(BMI)(P<0.05),and HDL disorder had association with tumor stage(P<0.05).4.The results of survival analysis showed PC patients with hyperlipemia has poorer prognosis than those with normal serum lipid,but there was no statistical difference.Thus,hyperlipemia was not the independent risk factors to affect the overall survival of PC patients after surgery.Conclusion:1.PC patients general suffer from hyperlipemia,which is mainly showed TG disorders.2.Hyperlipemia has closely relationship with clinicopathological features,including tumor location,BMI index and tumor grading.3.Pancreatic cancer patients with hyperlipemia have poor prognosis compared with patients with normal serum lipids,but there is no significant difference.It needs further to study through collecting multicenter and large sample size samples.Part 2 The mechanism study of high saturate fatty acid induces macrophages pyroptosis and promotes pancreatic cancer cells proliferation,migration and invasion through NLRP3/IL-1β axisObjective:To study the biologic behaviors and molecular mechanism of pancreatic cancer cells influenced by pyroptosis of macrophages and its inflammatory reaction via building high lipid microenvironment in vitro.Methods:1.High lipid microenvironment was built by palmitic acid in vitro,and macrophages were cultured in the condition.Flow cytometry was used to detect the phenotypes of macrophages.2.Changes of macrophages morphology was observed in inverted microscope,and cell membrane integrity was assessed by fluorescence microscope stained by SYTOX Green Nucleic Acid Stain.3.Expression of NLRP3 was detected by Western Blotting and qRT-PCR.Western Blotting was also used to detect the expression of inflammatory biomarkers(cleaved IL-1β、cleaved IL-18),pyroptosis biomarkers(Caspase-1,GSDMD-N)in protein level.ELISA was used to detect the release of IL-18 and IL-1β.LDH-Cytotoxicity Assay Kit was used to detect the release of LDH.Expression of NLRP3 was inhibited by CY-09,and related indicators was detected as above.4.Building the co-culture system of macrophages and PC cells,CY-09 inhibited the expression of NLRP3 of macrophages,and Canakinumab neutralized the IL-1β from macrophages.EdU cell proliferation assay,Wound Healing assay and Transwell assay were used to evaluate the biological behaviors of PC cells Change of Epithelial-mesenchymal transition(EMT)indicators were detected by Western Blotting and Immunofluorescence assay.Results:1.Stimulated by palmitic acid,the proportion of CD 8 6+macrophages significantly increased(P<0.01),and the phenotype polarizes into Ml.2.Stimulated by palmitic acid,macrophages showed plasmalemma swollen into a ball with pore formation,and changes into large bubbles,which was the sign of pyroptosis.Palmitic acid increased the permeability of plasma membrane,which was positive with stimulated time.3.Stimulated by palmitic acid,expression of NLRP3,cleaved IL-1β and cleaved IL-18 are increased,which then activated Caspase-1 and further induced N-terminal cleaved of GSDMD,accompanied by release of IL-1β,IL-18 and LDH.Inhibition of expression of NLRP3 in macrophages,expression of above indicators decreased,along with reduced release ofIL-1β,IL-18 and LDH.4.Conditioned medium of macrophages treated by palmitic acid enhanced the ability of proliferation,migration and invasion of PC cells,but above biological behaviors of tumor cells were attenuated by inhibitor of NLRP3,CY-09,or canakinumab,a neutralizing antibody of IL-1β.5.Conditioned medium of macrophages treated by palmitic acid inhibited expression of epithelial cell markers(E-cadherin),was not interstitial cell markers(Vimentin)in PC cells.But the expression of EMT indicators was the opposite(E-cadherin increased and Vimentin decreased)after inhibition of NLRP3 or neutralize IL-1β.Conclusion:1.High lipid microenvironment induced macrophages polarizing into proinflammatory M1 phenotype,and promoted macrophages pyroptosis via Caspase-1/GSDMD pathway,then released large of IL-1β mediated by NLRP3.The inflammatory response accelerated EMT,and further promoted migration and invasion of PC cells.