Heterogeneity Analysis Of Pancreatic Cancer And The Effect Of Pancreatic Astrocyte Glutamine Metabolism On Tumor Cells

Background:Pancreatic ductal adenocarcinoma(PDAC)is a malignancy featured with poor prognosis.Although considerable efforts have been made to identify biomarkers and subtypes at different levels in PDAC through high-throughput analyses,few of them have been recommended by the guidelines for routine oncology practice and the current conventional empiric therapy for PDAC was far from satisfactory,partly attributed to the underlying heterogeneity.Previous studies evaluating the heterogeneity in PDAC have mainly focused on genomes through whole-exome sequencing.However,a comprehensive investigation of PDAC heterogeneity through multi-region sampling at different dimensions is lacking.Methods:To comprehensively explore intratumoral and intertumoral heterogeneity in PDAC and evaluate the potential effects of heterogeneity on the pathogenesis,treatment and prognosis of PDAC,we analyzed 61 lesions and 19 matched control specimens from 19 PDAC patients by whole exome sequencing(WES),RNA-sequencing(RNA-seq)and methylated DNA Immunoprecipitation Sequencing(MeDIP-seq).Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to confirm gene mRNA expression levels.The protein expression levels of genes were evaluated by immunohistochemistry(IHC)staining.Results:Widespread intratumoral heterogeneity was present,though it was lesser in degree than intertumoral heterogeneity upon comprehensive viewing of genomes,transcriptomes,and epigenomes.Genomic analyses found that most intratumoral heterogeneity might be due to passenger gene mutations rather than driver gene mutations.However,a considerable number of spatial-and temporal intratumoral heterogeneity of mutations was also detected in driver genes,including major PDAC driver genes KRAS,CDKN2A,and TP53.Transcriptional profiling revealed that PDAC tumor microenvironment(TME)showed more intratumoral heterogeneity than in tumor cells.Moreover,we observed that high average copy number variation(CNV)burden was significantly correlated with liver metastasis(p=0.033),that chromosome 16p13.3 deletion(p=0.033)and low RAB11FIP3 expression(mRNA:p=0.0188;protein:p=0.0138)were significantly associated with shorter OS(overall survival).Conclusions:Our multi-omic integrative analyses provide a comprehensive view of molecular heterogeneity and evolutionary trajectories of PDAC,and demonstrated that high average CNV burden,chromosome 16p13.3 deletion and low RAB11FIP3 expression maybe potential prognostic factors of PDAC.In conclusion,our findings may support future decision making with respect to targeted-and immunotherapy strategies.Backgroud:Metabolic interventions are now emerging as potential therapeutic strategies for cancer treatment.However,the changes of metabolic characteristics(metabolic reprogramming)in the occurrence and development of the same tumor,which is one of the causes of the heterogeneity,poses a challenge to the development and utilization of the cancer therapies targeting to the metabolic characteristics.The glutamine metabolism reprogramming is occurred during the development of cancers,and then futher affects the biological characteristics of tumor cells.The metabolism of tumor microenvironment(TME)in pancreatic adenocarcinoma(PDAC)play a role in regulating the biological characteristics of pancreatic cancer cells,such as chemoresistance.Our first part of the study had already confirmed that the heterogeneity of TME is greater than that of pancreatic cancer cells(PCCs)in PDAC.In this part of the study,we focused on pancreatic stellate cells(PSCs),which is the main component of TME,to investigate the regulatory effect and mechanism of glutamine metabolism reprogramming of PSCs on the chemoresistance and other biological characteristics of PCCs.Methods:The effect of PSCs on PCCs proliferation and chemotherapy sensitivity was tested through PSCs conditioned medium,transwell and 3D co-culture system.Bioinformatics analyses were performed to screen out the key targets of PSCs glutamine metabolism pathway.The specific expression levels of key targets in PSCs and PCCs were compared by real-time PCR and western blot.Real-time PCR,western blot,and metabolomics profiling were used to explore the regulatory mechanisms of glutamine metabolism reprogramming in PSCs.Results:We found that PCCs were particularly dependent on the glutamine metabolism of PSCs in PDAC.We screened out the key targets of PSCs glutamine metabolism pathway was SLC1A2,which was a kind of glutamate transporter.The expression of SLC1A2 in PSCs was specifically higher than that in PCCs.Moreover,the findings showed that knockdown of SLC1A2 resulted in decreased activation levels of PSCs,and the expression levels of glutamine synthetase(GS)and the relative concentration of glutamine were decreased,suggesting the SLC1A2 in PSCs affected the glutamine metabolism reprogramming.Further ananyses demonstrated that knockdown of SLC1A2 in PSCs reduced the proliferation capacity of PCCs and promotion the sensitivity of PCCs to chemotherapy drug(Gemcitabine,GEM).Conclusions:SLC1A2 has an effect on the expression of GS in PSCs.And the activation and glutamine metabolism reprogramming of PSCs could also be regulated by SLC1A2,which further play a role in chemoresistance of PCCs through regulating the secretion of glutamine metabolites and other cytokines of PSCs.This study mainly explores the new mechanisms of PDAC chemoresistance from the perspective of metabolism in the tumor microenvironment,so as to provide the application value and efficient therapeutic targets for the treatment of PDAC.