The Effect And Mechanism Of Non-alcoholic Fatty Liver In The Occurance And Development Of Pancreatic Ductal Adenocarcinoma Liver Metastasis

Objective The etiology of pancreatic ductal adenocarcinoma liver metastasis(PDAC liver metastasis)is complex and the mechanism is unclear.In this study,we investigated the relationship between non-alcoholic fatty liver disease(NAFLD)and PDAC liver metastasis by constructing a non-alcoholic fatty liver disease-induced mouse model of pancreatic cancer liver metastasis.To evaluate the impact of NAFLD on the efficacy of chemotherapy in patients with pancreatic cancer liver metastases.Methods Wild-type mice aged approximately 8 weeks were selected and randomly divided into two groups.The experimental group was fed a high-fat choline-deficient L-amino acid diet(CDAA-HFD)for 4 weeks,while mice fed a normal diet were used as controls.A pancreatic cancer cell line(KPC)was injected into the spleen of mice with non-alcoholic fatty liver disease(NAFLD)and normal liver to establish a pancreatic cancer liver metastasis model.Two weeks later,the mice were killed and the liver metastases were recorded and quantified in terms of size and severity.The liver metastases were stained with H&E to observe the extent of liver metastasis and fibrosis.in order to analyse the effect of NAFLD on the microenvironmental fibrosis and immune infiltration in the liver metastases of pancreatic cancer tumours,immunohistochemical staining was performed to detect the expression of CD3,α-smooth muscle actin(α-SMA)in the metastases.Seventy-two patients with pancreatic cancer liver metastases treated with gemcitabine chemotherapy at the Affiliated Hospital of Qingdao University from March 2015 to March 2021 were enrolled to assess the effect of NAFLD on chemotherapy efficacy for pancreatic cancer liver metastases.Hepatic metastases from experimental and control mice were collected and subjected to proteome and transcriptome sequencing.The transcriptomic and proteomic sequencing data were compared with the transcriptomic molecular subtypes of pancreatic ductal adenocarcinoma by Bailey,Collisson,Moffitt et al.and the proteomic molecular subtypes of PDAC liver metastases by Henry to analyse the effects of NAFLD on molecular typing,prognostic stratification and chemotherapy sensitivity of PDAC liver metastases.To further explore the possible molecular mechanisms and intervention strategies,we downloaded single-cell sequencing data from the public database(The Gene Expression Omnibus,GEO)for pancreatic ductal adenocarcinoma and NAFLD,respectively.The PDAC and NAFLD single-cell sequencing data were combined for cell communication analysis to explore the interaction between PDAC cells and NAFLD cells.A mouse model of NAFLD driven PDAC liver metastasis was established and intervened with DMSO,MIF inhibitor ISO-1 or gemcitabine daily for one week prior to tumour cell injection for two weeks.Mice were sacrificed on postoperative day 15 and livers were dissected to assess the severity of liver metastases.H&E staining was performed on the liver metastases of the mice.Results Animal experiments revealed that mice with NAFLD had more severe liver metastases,significantly higher liver weight/body weight percentage(p<0.05),and poorer prognosis(p<0.05).H&E staining indicated that mice with NAFLD had larger liver metastases and significant fibrosis.Immunohistochemical results showed that α-SMA and Sirius scarlet staining were significantly enhanced in mice with NAFLD,but CD3 staining was weakened.Bioinformatic analysis showed a significant correlation between molecular subtypes of PDAC liver metastases driven by NAFLD and those identified by Moffitt et al.(p(tumour)= 0.083 and p(stroma)= 0.014),Collisson et al.(p=0.05)Bailey et al.(P=0.05)and Henry et al.(p=0.014).Clinical data suggest that PDAC liver metastasis patients with NAFLD are significantly more resistant to gemcitabine,with 70% reaching progressive disease(PD)within two cycles compared to 9.6% in patients without NAFLD(p<0.05).By cell communication analysis of single cell sequencing data from public databases,we found that the interaction of the ligand-receptor combination macrophage migration inhibitory factor(MIF)-CD74/CD44 was significantly enhanced.Administration of the MIF inhibitor ISO-1 prior to tumour cell injection significantly inhibited liver metastasis and attenuated fibrosis in the NAFLD group of mice.Conclusion 1.The mice in the NAFLD group exhibited extensive liver metastasis and poor prognosis.2.The degree of fibrosis in PDAC liver metastases was significantly enhanced and immune cell infiltration was reduced in mice with NAFLD.3.PDAC liver mrtastasis patients with NAFLD exhibit increased resistance to gemcitabine and poorer efficacy of chemotherapy.4.NAFLD group have molecular subtypes that are more similar to the squamous and inflammatory types.These subtypes are often associated with fibrosis,poor prognosis and resistance to chemotherapy.