| Pancreatic cancer,the most malignant tumors of the digestive system,has replaced liver cancer as the new "king of cancer",among which pancreatic ductal adenocarcinoma(PDAC)is one of the most aggressive solid malignancies.Due to the inherent tumor heterogeneity,high connective tissue hyperplasia,and immunosuppressive tumor microenvironment(TME)in PDAC,PD AC is resistant to chemotherapy,targeted therapy,and even immunotherapy,resulting in poor prognosis and high mortality in pancreatic cancer patients.Therefore,it is urgent to further reveal the molecular mechanism of the occurrence and development of pancreatic cancer,especially PD AC,and to explore new and effective targets for the diagnosis,treatment and prognosis of pancreatic cancer.The TME is closely related to the genesis,growth,metastasis and metabolism of tumors,in which dense connective tissue and immunosuppressive cell populations can be enriched in the TME of PD AC,limiting the cytotoxic T cell activity.Extracellular matrix proteins and cytokines produced by proliferating connective tissue and immunosuppressive TME interact with cancer cells to promote tumor growth and malignant progression.Recently,many therapeutic strategies targeting the tumor stromal microenvironment have increased tumor sensitivity to chemotherapy and/or immunotherapy,however,clinical trials on aggressive tumors have not yet yielded satisfactory results,and even some of these approaches have accelerated tumor progression.Given the importance of the tumor stromal microenvironment in the occurrence and development of PDAC,there is an urgent need to further understand the tumor stromal microenvironment and develop effective therapies for TME.Cancer stem cells(CSCs)have been shown to be closely related to tumor initiation,growth,metastasis,recurrence,and drug resistance in a variety of cancers,and recent studies have shown that CSCs are closely related to the innate immunosuppressive properties of tumor cells.The immune system plays an important role in the inhibition of malignant transformation of cells and tumor development.CSCs have an intrinsic mechanism to evade immune surveillance and thus escape the killing of immune cells.However,from an epigenetic perspective,little is known about how the interaction between CSCs and immune escape is regulated.In recent years,the expression of PDL1 in tumors has been recognized as a potential biomarker of the efficacy of immunotherapy,and blocking the PD-1/PD-L1 axis can enhance the T cell response,exhibiting significant clinical efficacy in a variety of advanced cancers,but in some patients,especially in PD AC patients,it does not produce a sustained anti-cancer effect.Therefore,immune-based strategies to combat this highly lethal malignancy are difficult to achieve satisfactory results.The in-depth study of the molecular mechanism of CSCs on the occurrence and development of pancreatic cancer and the induction of immune escape of pancreatic cancer has important guiding significance for the development of new targeted therapies for pancreatic cancer based on immunotherapy.In this study,chromatin accessibility assay ATAC-seq was used to analyze the changes of chromatin accessibility in pancreatic cancer at the genome scale.It was found that DDX18 was up-regulated in PD AC,which promoted the occurrence and development of PD AC and was verified at the epigenetic and transcriptomic levels.Its upregulation was confirmed to associate with poor prognosis in PDAC patients,.Based on these foundings,the regulatory role of DDX18 in PD AC was further explored in order to provide a new theoretical basis for the clinical treatment of PDAC patients.Research purpose(1)To explore the role of DDX18 in the occurrence and development of PDAC.(2)To explore the molecular mechanism by which DDX18 promotes PDAC stemness properties and thus immune escape.(3)To detect the role of DDX18 in anti-PD-L1 immunotherapy for pancreatic cancer.Part Ⅰ Chromatin remodeling occurs in pancreatic cancer resulting in elevated DDX18 expression[Object]In the field of oncology research,ATAC-seq technology is widely used in the study of tumor pathogenesis,drug action mechanism,new drug and biomarker development.In this part,the combination analysis of ATAC-seq and RNA-seq results is applied to find the key regulatory factors in the tumorigenation and development of PDAC,so as to explore the theoretical basis for establishing effective diagnostic biomarkers,developing new treatment schemes,and predicting or monitoring drug efficacy.[Methods and results]1.1 ATAC-seq data analysis revealed distictive chromatin remodeling in PDAC tumors and adjacent tissuesAfter unloaded the ATAC-seq raw data,data quality control was performed.Then conducted the reference genome comparison analysis,including fragment length distribution analysis,open chromatin site(Peak)identification,including peak length distribution analysis,PCA cluster analysis,open chromatin signal distribution analysis,peak motif analysis,peak function notes,peak target gene analysis.Analysis of ATACseq data showed that PDAC tumors and paracancer tissues underwent different chromatin remodeling,with most of the altered regions in tumor tissues compared to paracancer tissues located in promoter and intergenic regions where cis-regulatory elements,or enhancers,are located.GO/KEGG enrichment analysis showed that transcriptional regulation,DNA binding and RNA stabilization pathways were significantly enriched in tumor tissues compared with paracancer tissues in high-quality ATAC-seq libraries.1.2 Analysis of differentially expressed genes with RNA-seq dataRaw Reads of RNA-seq data were processed by removing low-quality sequences,joint contamination and ribosome sequences(rRNA)to obtain Clean Reads.All subsequent analyses were based on Clean Reads.Clean Reads were then compared to reference genes,and transcriptome expression was quantified from the comparison results.Differential genes were calculated by FPKM and genes with 2-fold change were screened.The differential genes were presented as volcano maps.1.3 The integrated analysis of ATAC-seq and RNA-seq data showed that DDX18 was highly expressed in pancreatic cancer tissues(1)After ATAC-seq analysis,the Difference Peak was obtained,and the Difference Peak was integrated with RNA-seq data to identify 9 genes with significant differences in expression between pancreatic cancer and adjacent tissues.(2)In the paired tumor tissues of PDAC patients,differential gene expression was verified by qRT-PCR and Western blotting.The results showed that DDX18 significantly increased in both mRNA and protein levels compared with the adjacent tissues.(3)ATAC-seq analysis showed that chromatin remodeling occurred in DDX18 promoter region in pancreatic cancer tissue and DDX18 expression was higher in pancreatic cancer tissue than in paracancer tissue in corresponding RNA-seq results.(4)The differentially expressed genes in tumor and paracancer tissues of PDAC patients was analyzed in the pancreatic cancer database,and the results showed that DDX18 was significantly higher in pancreatic cancer tissues than paracancer tissues.(5)Organoids derived from pancreatic tumor tissues and adjacent tissues of KPC mice were cultured,organoid protein samples were collected,and protein expression of DDX18 was detected by Western blotting.The results showed that the organoids derived from tumor tissues had rough surfaces and obvious invasion characteristics.The expression of DDX18 in tumor-derived organoids was significantly higher than that in para-cancerous organoids.(6)Immunofluorescence(IF)and immunohistochemistry(IHC)stainings were used to detect the protein expression of differentially expressed genes in tumor tissues and adjacent tissues of KPC mice and pancreatic cancer patients,and the results showed that DDX18 expression was significantly higher in pancreatic cancer tissues than in adjacent tissues.(7)ChIP-qPCR was used to detect the enrichment of histone activation markers H3K27ac and H3K4me3 in the DDX18 promoter region.The results showed that H3K27ac and H3K4me3 were significantly enriched in the promoter of DDX18 in pancreatic cancer tissue and organoids derived from tumor tissue.(8)The relationship between DDX18 expression level and patient survival was analyzed using the pancreatic cancer database.The results showed that the high expression of DDX18 was negatively correlated with the prognosis of patients.(9)Commercial pancreatic cancer tissue chip was used to analyze the relationship between DDX18 expression level and patient survival.The results showed that the high expression of DDX18 was negatively correlated with the prognosis of patients.[Conclusion]The characteristic remodeling of chromatin in pancreatic cancer tissues leads to increased DDX18 expression,which is not conducive to the prognosis of patients.Part Ⅱ DDX18 promotes the development of pancreatic cancer[Object]In the first part,we found that the elevated expression of DDX18 in tissues of PD AC patients is negatively correlated with the prognosis of patients.Next,we will explore the role of DDX18 in the occurrence and development of PD AC through in vivo and in vitro experiments.[Methods and results]2.1 DDX18 promotes the proliferation and metastasis of pancreatic cancer cells in vitro(1)Western blotting detected the basal expression level of DDX18 in PD AC cell lines,and based on this,lentivirus-stable human and murine DDX18 high expression and silent pancreatic cancer cell lines were constructed.(2)EdU staining,clonal formation,and MTS experiments were conducted using the stable cell line,and the results showed that DDX18 promoted the proliferation of PDAC cells.(3)Scratch,Transwell and Matrigel experiments were conducted using the stable cell lines,and the results showed that DDX18 promoted the migration and invasion of PDAC cells.2.2 DDX18 promotes growth and metastasis of pancreatic cancer in vivo(1)The constructed stable cell lines were subcutaneously inoculated into the nude mice and C57/B6 mice,and the tumor growth rate and size were evaluated by tracking and measuring tumor volume and weight.The results showed that DDX18 promoted the occurrence and growth of PDAC tumors.(2)The above-mentioned stable transmissible cell lines were injected into mice through the tail vein,and the lungs were dissected,weighed,fixed,embedded,sectored,stained,and metastases counted.It was found that DDX18 promoted the metastasis of PDAC cells in vivo.[Conclusion]In vitro and in vivo experiments showed that DDX18 can promote the development of pancreatic cancer.Part Ⅲ DDX18 can enhance the stem cell properties of pancreatic cancer[Objective]In the second part,we confirmed that DDX18 can promote the occurrence and development of PDAC through in vivo and in vitro experiments.Next,we will further explore the specific ways of DDX18 promoting malignant progression of PDAC.[Methods and results]3.1 DDX18 enhanced the stemness properties of pancreatic cancer in vitro(1)Western blotting detected the protein expression levels of stemness characteristic markers in cell lines with high DDX18 expression and silenced DDX18 expression and the results showed that the expression levels of DDX18 were significantly positively correlated with the expression of stemness markers.(2)Flow cytometry was used to detect the proportion of CD24+CD44+double positive cells(CSCs)in DDX18 high expression and silent cell lines.The results showed that DDX18 increased the proportion of stem cells,indicating that DDX18 could enhance the stemness of PDAC cells.(3)The tumor sphere formation ability of cells with high expression and silence of DDX18 was detected in the low adsorption culture plate by serial sphere formation experiment.The results showed that DDX18 promoted the formation of serial sphere,indicating that DDX18 could enhance the stemness of PD AC cells.(4)The effect of DDX18 on the growth ability of organoids derived from mice of KPC pancreatic cancer was tested by organoid culture.The results showed that DDX18 promoted the formation and growth of organoids,indicating a significant positive correlation between DDX18 and PD AC stemness characteristics.(5)Mouse pancreatic cancer organoids with high and low DDX18 expression were collected,and the effects of DDX18 on the expression level of stemness markers in the organoids were detected by Western blotting.The results showed that the expression level of DDX18 was significantly positively correlated with the expression of stemness markers.(6)The mouse pancreatic cancer organoids with high expression and silence of DDX18 were tracked and recorded,and the size and number of organoids were statistically analyzed.The results showed that DDX18 significantly promoted the growth of organoids.3.2 DDX18 enhances pancreatic cancer stem cell properties and promotes PD AC formation and growth in vivoLimited dilution of DDX18 silenced PD AC cell lines were subcutaneously inoculated into mice.The results of in vivo experiments showed that DDX18 silencing significantly reduced the proportion of CSC in PDAC cell lines,and the decrease of DDX18 expression level seriously affected the formation and growth of PD AC tumors.[Conclusion]DDX18 enhances the stem cell properties of pancreatic cancer in vivo and in vitro,and promotes the occurrence and development of pancreatic cancer.Part Ⅳ DDX18 promotes the expression of STAT1 by antagonizing the formation of PRC2 complex[Object]In the third part,we tested and verified that DDX18 can promote the malignant progression of PDAC by enhancing the stemness characteristics of PDAC cells through various experimental methods.In the following part,we want to further explore the molecular mechanism causing this malignant progression.[Methods and results]4.1 CUT&Tag combined RNA-seq analysis was used to search for DDX18-related downstream signaling pathways(1)RNA-seq analysis was performed in DDX18 highly expressed and control cells,and the data analysis results showed that differentially expressed genes were distributed in many functional pathways,mainly enriched in transcriptional regulation and immune-related signaling pathways.(2)CUT&Tag experiment were carried out in the same cells above,and the analysis of experimental results showed that differentially expressed genes corresponding to differentially expressed peaks were distributed in many functional pathways,mainlyenriched in cell development and immune microenvironment pathways.(3)Gene enrichment analysis(GSEA)was performed with CUT&Tag combined with RNA-seq results:the results showed higher scores in immune-related and stemness-related signaling pathways,with significant statistical significance.4.2 The verification of the omics analysis donfirmed that STAT1 expression was significantly positively correlated with DDX18 level(1)qRT-PCR was used to detect the expression level of the enriched differential genes in PD AC cells with stable high and silent expression of DDX18.The results showed that STAT1 and STAT3 were significantly affected by DDX18 expression.(2)Western blotting was used to detect the protein levels of STAT1 and STAT3 in PD AC cells with stable high and silent expression of DDX18.The results showed that only the protein expression of STAT1 was significantly affected by DDX18.(3)Western blotting experiment was used to detect DDX18 and STAT1 protein level in pairs of pancreatic cancer and adjacent tissues,and the results showed a significant correlation between their expressions.(4)Public database analysis of DDX18 and STA T1 expression showed that there was a significant correlation between their expression level.(5)IHC was used to detect the protein levels of differential genes in subcutaneous tumor tissues of mice with high and silent DDX18 expression,and the results showed that the expression of STAT1 was significantly positively correlated with DDX18 expression.4.3 DDX18 promotes the expression of STAT1 at the transcription level(1)Integrative Genomics Viewer will be used to obtain the signal distribution of Peak in STAT1 and STAT3 promoter regions and conduct statistical analysis.The results show that the signal in STAT1 promoter region was significantly affected by DDX18 expression level.(2)The accumulation of DDX18 in the STAT1 promoter region and the influence of DDX18 on the aggregation of H3K27me3 in the STAT1 promoter region were detected by ChIP-qPCR.The results showed that DDX18 was significantly enriched in the STAT1 promoter region and could affect the deposition of H3K27me3 in the STAT1 promoter region.(3)The effect of DDX18 on the transcriptional regulation of STAT1 was detected by dual luciferase assay,and the results showed that DDX18 could promote the expression of STAT1 at the transcriptional level.4.4 DDX18 restrains the formation of PRC2 complex by antagonizing the binding of EZH2 to other components of PRC2,and reduces the enrichment of H3K27me3 in STAT1 promoter region(1)Co-immunoprecipitation(Co-IP)in PD AC cells with high DDX18 expression showed that DDX18 could bind SUZ12 and EED,but not EZH2.After DDX18 overexpression,the binding of SUZ12 and EED to DDX18 increased,but the binding of EED to EZH2 decreased.Co-IP experiments using EZH2 antibodies showed that EZH2 did not bind to DDX18,and binding to SUZ12 and EED was reduced.The results showed that DDX18 directly interacts with the PRC2 complex and inhibits the binding of EZH2 to other components of the PRC2 complex.(2)Co-IP in DDX18-silenced PD AC cells showed that SUZ12 and EED antibodies were able to capture DDX18,nearly the same amount of EED/SUZ12,and more EZH2,and EZH2 binding to SUZ12 and EED increased significantly after silencing DDX18.The results indicated that the loss of DDX18 expression enhanced the binding of EZH2 to other PRC2 components.(3)EZH2 was knocked down in DDX18-silenced cells,and Co-IP experiments were performed using SUZ12 and EED antibodies.The results showed that after silent EZH2 expression,the binding of SUZ12 and EED to DDX18 was enhanced.(4)Knocking down EZH2 in DDX18-silenced cells and then performing ChIP experiments for H3K27me3 showed that knocking down EZH2 reduced H3K27me3 enrichment in the STAT1 promoter region of DDX18-silenced cells.[Conclusion]By antagonizing EZH2 binding to other components of PRC2 complex,DDX 18 inhibits the formation of PRC2 complex,reduces the accumulation of H3K27me3 in the STAT1 promoter region,and promotes the expression of STAT1 at the transcriptional level.Part V ST AT1 mediates the function of DDX18 in maintaining the sternness and immune escape of PD AC cells[Object]In the fourth part,we enriched the differentially expressed genes and pathways in PD AC through data integration analysis,and conducted an in-depth exploration on how DDX18 affects STAT1 expression.Next,we would like to further explore whether DDX18 exerts its function in pancreatic cancer through ST AT1.[Methods and results]5.1 DDX18 enhanced PDAC stem cell characteristics,induced PDAC immune escape,and promoted the development of PD AC5.1.1 In orthotopic pancreatic cancer mouse model demonstrated that DDX18 promotes malignant progression of pancreatic cancer(1)KPC pancreatic cancer orthotopic models with high expression and silence of DDX18 were constructed and randomly divided into 2 groups.One group was used to evaluate the effect of DDX18 on tumor growth,and the other group was used to track and evaluate the effect of DDX18 on the survival of the model.The results showed that high DDX18 expression significantly promoted the growth of tumors,while low DDX18 expression significantly inhibited the growth of tumors.The survival statistics showed that the high expression of DDX18 was not conducive to the survival of model mice.(2)HE staining of the above tumor tissue indicated that DDX18 accelerated the malignant progression of the tumor.(3)In orthotopic mouse models,significant liver metastasis was found in the group with high DDX18 expression,but not in the group with silent DDX18,indicating that DDX18 promoted the metastasis of pancreatic cancer.5.1.2 DDX18 maintains the stemness properties of PDACThe above fresh KPC mouse tumor tissues were used for organoid culture.The results showed that DDX18 could maintain the stemness characteristics of PDAC and promote the formation and growth of organoids.5.1.3 DDX18 induces immune escape of PDAC(1)IF staining and flow cytometry were used to detect the infiltrated T cells in the above tumor tissues,and the results showed that DDX18 could promote the local infiltration of T cells at the tumor site.(2)ELISA and flow cytometry were used to detect the expression of T cell depletion markers in the above tumor tissues,and the results showed that DDX18 could significantly increase the expression of T cell depletion markers.(3)The above tumor tissues were stained with Sirius red,and the results showed that DDX18 could significantly increase the deposition of collagen in TME and produce immunosuppressive TME.(4)In orthotopic pancreatic cancer tissues with high DDX18 expression,Western blotting detected the protein expressions of DDX18,STAT1,PD-L1 and EMT markers,and the results showed that the protein expressions of STAT1,PD-L1 and EMT markers were significantly positively correlated with the expression levels of DDX18.5.2 The role of STAT1 in the occurrence and development of PD AC5.2.1 ST AT1 promotes the development of PD AC(1)Mouse Pan02 pancreatic cancer cell line with high and silent expression of STAT1 was constructed using lentivirus system.Ki67 staining showed that the expression of ST AT1 was significantly positively correlated with the proliferation ability of pancreatic cancer cells,and Transwell and Matrigel experiments showed that STAT1 could promote the migration and invasion of PD AC cells.(2)Pan02 cell line with stable STAT1 expression was inoculated subcutaneously into C57/B6 mice,and the effect of STAT1 on tumor growth was evaluated by measuring tumor volume.Tumor volume and weight were measured 28 days after tumor implantation.The results showed that STAT1 significantly promoted the development of PDAC tumors in vivo.(3)KPC pancreatic cancer mouse models with STAT1 silencing was constructed,and the mice were randomly divided into two groups,one group was used to detect the effect of STAT1 on tumor growth,and the other group was used to track and evaluate the effect of STAT1 on the survival of the mouse model.The results showed that tumor growth was significantly inhibited in the STAT1 silent group compared with the control group.Pathological staining of tumor tissue showed that STAT1 silence could prevent the further deterioration of tumor.Survival statistics showed that STAT1 silence effectively prolonged the survival of pancreatic cancer mouse models.(4)HE staining was performed on KPC mice pancreatic cancer tissues with STAT1 silence,and the results showed that STAT1 silence could inhibit the malignant progression of pancreatic cancer.5.2.2 STA T1 maintains the stemness properties of PD AC(1)PDAC cell lines with stable and high STAT1 expression were used for serial sphere formation experiment and the results showed that STAT1 could maintain the stemness characteristics of PDAC.(2)Flow cytometry was used to detect the effect of STAT1 on the proportion of CSCs in the subcutaneous tumor tissues of the above mice,and the results showed that the expression level of STAT1 was closely related to the CSC proportion of PDAC.(3)KPC murine-derived pancreatic organoids with high and low STAT1 expression were constructed using the lentivirus system,and the effects of ST AT1 on the characteristics of pancreatic CSCs were evaluated.The results showed that high STAT1 expression could promote the formation and growth of organoids.(4)Limited dilution of STAT1 overexpressed Pan02 cell lines were inoculated into C57/B6 mice,and the results showed that STAT1 could increase the proportion of CSCs in vivo.(5)It is found that STAT1 silence inhibits the stemness characteristics of PDAC and thus inhibits the formation and growth of organoids by culturing organoid from fresh KPC pancreatic cancer tissues with ST AT1 silence.5.2.3 ST AT1 induces immune escape of PDAC(1)Western blotting detected the protein expression of PD-L1 and STAT1 in cell lines with high and silent STAT1 expression,and the results showed a significant positive correlation between their expression.(2)The IHC detected the expression of PD-L1 in subcutaneous tumor tissues with high and silent STAT1 expression,and the results showed that the expression of PDL1 in tumor tissues was significantly positively correlated with the expression level of STAT1.(3)The effect of STAT1 on T cell infiltration in subcutaneous tumor tissue was detected by IF staining,and the results showed that STAT1 significantly increased the local infiltration of T cells in tumor.(4)Flow cytometry was used to detect the effect of STAT1 on the proportion of T cell infiltration and the expression of T cell depletion marker.The results showed that STAT1 promoted the local infiltration of T cell in tumor and the expression of T cell depletion marker.(5)ELISA assay was used to detect the expression of T cell depletion markers in tumor infiltration,and the results showed that STAT1 could promote the expression of T depletion marker and promote T cell dysfunction.(6)The effect of STAT1 on collagen deposition in intratumoral matrix was detected by Sirius red staining.The results showed that ST AT1 could significantly increase collagen deposition in TME and enhance the immune inhibition of TME.(7)IF staining and flow cytometry were performed on orthotopic tumor tissues silenced with STAT1.The results showed that STAT1 silencing effectively reduced T cell infiltration in tumor tissue and decreased the expression of T cell depletion markers.(8)Sirius red staining was performed in STAT1 silent orthotopic pancreatic cancer tissues,and the results showed that ST AT1 silencing reduced collagen deposition in TME and effectively improved TME.5.3 DDX18 maintains the stemness properties and promotes immune escape of PD AC through STAT15.3.1 In vitro experiments confirmed that DDX18 maintained PDAC stem cell properties through STAT1 and regulated PD-L1 expression(1)The CD24+CD44+double-positive PDAC CSCs in the organoids were sorted out by organoid culture and flow sorting techniques,and the protein expression levels of DDX18,STAT1,stemness characteristic markers and epithelial mesenchymal transformation(EMT)markers were detected by Western blotting.The results showed that the protein expression levels of DDX18,STAT1,stemness characteristic marker and EMT marker in PDAC stem cell protein samples were significantly higher than those in control group.(2)The expression of STAT1 was silenced in PDAC cell lines with high DDX18 expression.Western blotting results showed that the increased expressions of PD-L1,stemness characteristic markers and EMT marker proteins caused by DDX18 overexpression were reversed by STAT1 silencing.(3)STAT1 expression was silenced in the Pan02 cell line with high DDX18 expression,and STAT1 expression was overexpressed in the Pan02 cell line with DDX18 silence,and the above cells were inoculated into the low adsorption cell culture plate for serial tumor sphere formation experiments.Statistical analysis showed that the ability of serial sphere formation promoted by high DDX 18 expression was reversed by silencing of STAT1.The inhibition of DDX18 silence to serial sphere formation was enhanced by the high expression of ST AT1.The results showed that DDX18-STAT1 axis significantly promoted the stemness characteristics of PD AC.(4)STAT1 was silenced and overexpressed in the organoids derived from DDX18 high and silencing orthotopic mouse pancreatic cancer models.The results showed that the promotion of organoid growth caused by DDX18 high expression was reversed by STAT1 silencing,and the inhibition of organoid growth caused by DDX18 silence was reversed by STAT1 high expression.(5)DDX18 was enhanced in organoids drived from STAT1 silent orthotopic mouse pancreatic cancer model.The results showed that organoid growth inhibition induced by STAT1 silence was restored by overexpression of DDX18.5.3.2 In vivo experiments confirmed that DDX18 maintained PDAC stemness properties and promoted PD AC immune escape through STAT1(1)Pan02 cell lines with high DDX18 expression were silenced STAT1and Pan02 cell lines with DDX18 silence were enhanced STAT1 expression.The above Pan02 cell lines were inoculated subcutaneously into C57/B6 mice,and the tumor volume changes were continuously measured and recorded.28 days later,the tumor bodies were removed for measurement and weighing.The results showed that DDX18-STAT1 axis significantly promoted the occurrence and development of PDAC.(2)Western blotting and IFC detected the expression of PD-L1 and stemness markers in the above tumor tissues,and the results showed that the increase of the above protein expression caused by the high expression of DDX18 was reversed by the knockdown of STAT1;The decrease in the above protein expression due to DDX18 silence was reversed by the high expression of STAT1.(3)Flow cytometry detected T lymphocytes in the tumor tissues above,and the results showed that DDX18-STAT1 axis significantly promoted T cell infiltration in PDAC tumors.[Conclusion]DDX18 enhanced the properties of pancreatic cancer stem cells through STAT1 and formed immunosuppressive TME,thus promoting the immune escape of PDAC and the occurrence and development of PDAC.Part Ⅵ Targeted inhibition of DDX18 expression promotes the sensitive to antiPD-L1 immunotherapy in PDAC[Object]In the fifth part,we confirmed the important role of DDX18-STAT1 in pancreatic cancer through various experimental methods.In this part,targeting inhibition of DDX18 expression combined with anti-PD-L1 immunotherapy was used to treat pancreatic cancer model mice,and the role of DDX18 in PD AC immunotherapy was analyzed.[Methods and results]6.1 Targeted inhibition of DDX18 combined with anti-PD-L1 immunotherapy significantly inhibited the growth of subcutaneous tumors in PD AC(1)Pan02 cell lines with overexpressed and silent DDX18 expression were subcutaneously inoculated into C57/B6 mice,and one of the groups was randomly assigned to receive anti-PD-L1 immunotherapy,and the changes of tumors were tracked and measured.Statistical results showed that the DDX18 high expression strongly tolerated anti-PD-L1 immunotherapy.DDX18 knockdown is more sensitive to anti-PD-L1 immunotherapy.(2)The proportion of CSCs in subcutaneous tumor tissues with DDX18 silence was detected by flow cytometry.The results showed that the proportion of CSCs in tumor tissues was significantly reduced after combined treatment.6.2 The orthotopic DDX18 silent PD AC was significantly inhibited after anti-PDL1 immunotherapy(1)The established DDX18 silent mouse model of orthotopic pancreatic cancer was randomly divided into 2 groups,one of which was given anti-PD-L1 immunotherapy.The results showed that the growth of orthotopic pancreatic tumor was inhibited after immunotherapy,and the survival period of mice was effectively prolonged.(2)B-ultrasound was used to detect orthotopic pancreatic tumors in vivo,and the results showed that inhibition of DDX18 expression combined with anti-PD-L1 immunotherapy significantly inhibited tumor growth.(3)HE staining was used to detect the pathological changes of cancer tissues in the DDX18 silent orthotopic pancreatic cancer after anti-PD-Ll immunotherapy,and the results showed that after anti-PD-L1 immunotherapy,pancreatic tumor lesions were significantly reduced and the degree of pathological deterioration of tumors was weakened.(4)Sirius red staining showed that collagen deposition in tumor stroma was significantly reduced after anti-PD-L1 immunotherapy in DDX18 silenct orthotopic pancreatic cancer model.(5)Flow cytometry showed that the proportion of CSCs,the proportion of T cell infiltration and the expression of T cell depletion markers were significantly decreased in the DDX18 silent orthotopic pancreatic cancer model after anti-PD-L1 immunotherapy.(6)The IF staining of DDX18 silent orthotopic pancreatic cancer model showed that after treated with anti-PD-L1 immunotherapy,and T cell infiltration at the tumor site was reduced.(7)Organoids derived from DDX18 silent orthotopic pancreatic cancer tissue after anti-PD-L1 immunotherapy were cultured,and the results showed that organoid growth was significantly inhibited.[Conclusion]Inhibiting DDX18 expression in PDAC makes PDAC more sensitive to PD-L1 immunotherapy. |
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