The Study On Effect And Molecular Mechanism Of Pancreatic Stellate Cells On Proliferation In Pancreatic Cancer Through GS-Mediated Glutamine Metabolism

Background and Objective:Pancreatic cancer is one of the cancers with the worst prognosis,and the 5-year survival rate of pancreatic cancer is only 10%.Pancreatic ductal adenocarcinoma(PDAC)is the dominant histological type of pancreatic exocrine malignancies.Surgery is still the only effective treatment for PD AC,but less than 20%of patients have the opportunity for surgical resection.For patients with advanced pancreatic cancer,the efficacy of chemotherapy,radiotherapy and targeted therapy are very limited.The characteristic feature of PDAC is its distinctive tumor microenvironment(TME),which is composed of dense and abundant fibrous stroma.Activated pancreatic stellate cells(PSCs)are the main cell type in TME,which secrete extracellular matrix such as collagen and hyaluronic acid and contribute to the formation of desmoplasia in PDAC.On the other hand,PSCs exhibit continual crosstalk and co-evolve with pancreatic cancer cells(PCCs),becoming an essential component of their physiology,and contributing to tumor progression,metabolism and chemoresistance in PDAC.Growing evidence demonstrates that targeting PSCs is emerging as an attractive and viable therapeutic strategy to disrupt the interaction between stroma and PCCs.Based on an in-depth understanding of metabolic regulation,targeting tumor metabolism has made breakthroughs in treatment and prognosis prediction.PDAC cells exhibit a distinct metabolism,which is manifested by metabolic reprogramming between tumor cells and stromal cells,and increased uptake and dependence on extracellular glutamine(Gln).However,the roles and mechanisms of PSCs in Gln metabolic pathways and tumor-stromal metabolic crosstalk have not been well clarified in PDAC.The aim of this study was to investigate the effect and molecular mechanism of PSCs on PCCs proliferation through GS-mediated glutamine metabolism.Methods:In this study,[U-13C]-Gln injection was performed in an orthotopic PDAC mouse model,and qualitative flux analysis with isotope tracer was performed to analyze Gln uptake by tumor tissues.Using PDAC single-cell RNA sequencing(scRNA-seq)datasets,quantitative real-time PCR and western blot,we analyzed the transcription and protein expression levels of key enzymes of the Gln metabolic pathway,and the metabolic heterogeneity between different cell types in TME,especially in tumor cells and stromal fibroblasts.The expression of glutamine synthetase(GS)in tumor and stroma and the association of GS expression with clinicopathological characteristics in PDAC patients was evaluated by PDAC tissue microarrays,immunohistochemistry(IHC)staining and multiplexed immunofluorescence(mIF).The effect of Gln concentration on the proliferation of PSCs and PCCs cells was assessed by clonal formation assay.The effect of PSCs on PCCs metabolism was evaluated by measurement of oxygen consumption rate.Metabolomics was used to determine the metabolites provided by PSCs to PCCs.After transfected with small interfering RNAs(siRNAs)to inhibit GS expression in PSCs,GSmediated tumor proliferation in the PSCs-PCCs co-culture model was detected by CCK-8 assay.The mechanism of GS-mediated PSCs promoting tumor proliferation was evaluated by Gln synthesis and secretion detection,NADPH/NADP+ratio and cell cycle analysis.The tumor-promoting effect of GS in the orthotopic PDAC mouse model was evaluated by ultrasound and IHC.Moreover,using the database to predict the potential binding sites of the Wnt/β-catenin pathway and GS,we investigated the underlying regulatory mechanisms for GS in PSCs.Chromatin immunoprecipitation(ChIP),luciferase reporter assays,immunofluorescence staining and rescue assays were used to explore Wnt/β-catenin/TC7binding site within the GS promoter region and to clarify the molecular mechanism of PSCs promoting PCCs proliferation through Wnt/β-catenin-mediated Gln metabolism.Results:After[U-13C]-Gln injection was performed in the orthotopic PDAC mouse model,the incorporation of 13C-Gln increased rapidly.In contrast,the 13C-Gln levels were lower in the tumor tissues than in the corresponding normal adjacent tissues.In addition,we found that the level of Gln and tricarboxylic acid(TCA)cycle intermediates exhibited a downward trend in the tumor core regions compared with the normal adjacent tissues.PDAC single-cell RNA sequencing datasets analysis showed that the key enzymes involved in Gln metabolism,including GS,glutamic oxaloacetic transaminase 2 and glutamate dehydrogenase exhibited significantly higher expression levels in stromal cells compared to cancer cells.IHC staining in PDAC tissue samples showed that stromal fibroblasts,including PSCs,were the main cell types expressing GS.These results were further confirmed at mRNA and protein levels in PDAC-derived PSCs and PCCs.Based on IHC analysis,stromal cells had considerably higher expression of GS than tumor cells(P<0.001).High GS expression in either tumor or stroma was more frequent in PDAC patients with poorly differentiated tumors,respectively(P<0.01;P=0.015).A higher level of GS in the stroma was significantly correlated with the presence of lymphovascular invasion and T1-T2 stage(P=0.003;P=0.034).Kaplan-Meier analysis showed that patients with high expression of GS in the tumor and stromal compartments had poor survival outcomes(P<0.05).Univariate and multivariate analysis showed that high GS expression in tumor and stroma served as an independent prognostic factor for poor prognosis in PDAC patients(P<0.05).Co-expression of GS and the PSCs marker smooth muscle actin was assessed by mIF staining.In experiments,Gln depletion on cell Iviability and noticed that MIA PaCa-2 or PANC-1 colonies were hardly detectable in Glnfree medium.However,Gln deprivation had only modest effects on the growth and viability of PSCs.In addition,the measurement of oxygen consumption rate(OCR)showed that metabolites secreted by PSCs increased the basal OCR of PCCs.Metabonomic experiment further confirmed Gln was produced by PSCs and utilized by PCCs.GS silencing suppressed the Gln concentration in the supernatant of PSCs and intracellular Gln levels in PCCs.The NADPH/NADP+ratio was decreased in PSCs and PCCs with GS depletion.Upon the inhibition of GS expression,the cell growth rate of PCCs was significantly lower,and the G1 phase in PCCs was increased.In in vivo experiments,the stable knockdown PSCs were conducted by infecting the cells with lentiviruses encoding the shRNA targets to GS.We found that GS depletion in PSCs significantly decreased the tumor volume,weight and Ki-67 proliferation index.Mechanistically,transfection of PSCs with β-catenin siRNA led to reduced expression of GS.Conversely,the Wnt/β-catenin signaling pathway could be activated by Wnt3a treatment,leading to nuclear localization of β-catenin and elevated cytoplasmic GS expression.Using ChIP and luciferase reporter assays,TCF7 was identified as the transcriptional co-activator of β-catenin and directly bound to the GS promoter,leading to the upregulation of GS expression in PSCs.Rescue experiments showed that β-catenin knockdown significantly inhibited the expression of GS and Gln synthesis in PSCs,and suppressed the proliferation-promoting effects of PSCs on PCCs.Overexpression of GS reversed the decrease in the GS expression,Gln synthesis capacity and the tumorpromoting effects of PSCs caused by β-catenin knockdown.Conclusions:An in-depth understanding of metabolic interaction between stromal and tumor cells contributes to an attractive and viable therapeutic strategy.In this study,the metabolic heterogeneity of tumor cells and stromal fibroblasts was investigated,and key enzymes involved in Gln metabolism were highly expressed in PSCs,especially GS.High GS expression in tumor and stroma served as an independent prognostic factor for poor prognosis in PDAC patients.PSCs promoted the proliferation of PCCs by upregulating Gln synthesis and secretion,NADPH/NADP+ratio and cell cycle.The Wnt/β-catenin pathway upregulated GS expression through directly binding to the GS promoter region.Wnt/β-catenin/TCF7 signaling axis is responsible for the maintenance of Gln levels via the upregulation of GS expression in PSCs,in turn assisting tumor cells in overcoming nutritional stress.Our study provides new insights into the roles and mechanisms of stromal Gln metabolic pathways in PDAC,which may have important implications for novel therapeutic strategies.