Metabolic Features And Early Diagnosis Of Small Cell Lung Cancer Based On Next-generation Metabolomics

Objective: Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world.Small cell lung cancer(SCLC)accounts for about 10%-15% of lung cancer,with high malignancy.Most patients with SCLC are diagnosed at advanced stage and have a poor prognosis.The low detection efficiency of early screening is one of the main causes of high mortality.Chemotherapy is the mainstay of drug therapy in SCLC,but it is easy to be drug-resistant.Due to the genome instability and high mutability,the heterogeneity of SCLC is very high.Currently there are no effective molecular targeted drugs for clinical application.It is urgently needed to look for new targets that can be used for early diagnosis and treatment of SCLC to improve the survival status of patients.The change in cell metabolism is a hallmark event of cancer incidence,and this acknowledge has become the consensus of academic community.Cancer cells generally reprogram the existing metabolic pathways to support cell survival under stress or allow cells to grow and proliferate at pathologically elevated levels.Next-generation metabolomics is a new technology of systems biology developed in the post-genomic era,analysis of large-scale metabolites using metabolomics has become one of the new hot spots in studying cancer pathological mechanisms,evaluation of therapeutic effect and exploring biomarkers for early diagnosis.In recent years,due to the improved sensitivity,accuracy and bioinformatics analysis,the next-generation metabolomic techniques based on high performance liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS)have the capacity to simultaneously quantify the key small molecule metabolites of all the major metabolic pathways,which can directly and accurately reflect the current status of organisms,and allows us to comprehensively assess the metabolic abnormalities in cancer.Metabolic features of many types of cancers,including the lung cancer,have been reported,but it has not yet been fully explained.In recent years,more and more attention has been paid to the metabolism of lung cancer,however,the research on lung cancer by using metabolomics mainly focuses on non-small cell lung cancer(NSCLC),and few studies on the metabolic features of SCLC have been done.The purpose of this study was to elucidate the unique metabolic features of SCLC and to explore its pathogenesis in search of molecular targets of drugs and biomarkers for early diagnosis.Research methods: 1.The first part of this study.NCI-H446 human small cell lung cancer cells were selected as the experimental group and BEAS-2B human bronchial epithelial cells(HBEC)as control group.The metabolites was detected and analyzed using the next-generation targeted metabolomics based on LC-MS / MS after they were extracted.A total of 420 major metabolites were detected,covering 61 human main metabolic pathways.Multiquant 3.0 software,online data analysis tools of metabolomics,such as metaboanalyst,were used for bioinformatics data analysis.Key rate limiting proteins,chosen from metabolic pathways with significant differences,were analyzed by targeted proteomics and gene expression analysis(quantitative real-time PCR)to verify the findings of metabolimics.2.The second part of this study.The whole blood samples were taken from 37 newly diagnosed SCLC patients(experimental group)and 37 healthy volunteers(control group)who meet the inclusion criteria using dry blood spot tehcnqiue.The metabolites was detected and analyzed using LC-MS/MS based next-generation metabolomics after they were extracted.Statistical analysis was performed on male and female groups respectively.Fisher's exact test was used to compare the basic information of the participants.Multiquant 3.0 software,online data analysis tools of metabolomics,such as metaboanalyst,were used for bioinformatics data analysis,and for screening metabolic tumor markers in both male and female SCLC patients.Results: Part one: 1.Metabolomic analysis showed the dramatic metabolic differences between HBEC and SCLC cells.2.Large-scale correlation analysis revealed the significant reduction of metabolites-metabolites correlations(MMCs)in SCLC cells compared with HBEC.3.Metabolic pathway analysis revealed that glycolysis,TCA cycle,urea cycle,purine and pyrimidine metabolism,fatty acid oxidation and glycerophospholipid are the main disturbed pathways in SCLC cells.4.The expression of 6 key rate limiting proteins in the disturbed metabolic pathways including XDH,HPRT,APRT,CPS1,CPT1 A and CPT2,was significantly altered in SCLC cells(p<0.05)except CPT2,which had been verified by targeted proteomics.5.qPCR analysis showed that the expression of key genes of purine salvage pathway,including APRT,HPRT1 and XDH,were significantly reduced by 2-15 times than the control group.In contrast,CPS1(urea cycle)and CPT1A(fatty acid oxidation pathway)were overexpressed by 4-18 fold.6.The metabolic distrubances of purine salvage pathways,fatty acid oxidation and urea cycle pathways were confirmed by targeted proteomics and qPCR analysis.Integrated network analysis showed that purine salvage pathway,fatty acid oxidation and urea cycle are interrelated through mitochondrial metabolism.Part two: 1.Multivariate PLS-DA analysis revealed that the whole blood metabolic features of patients with SCLC in both men and women are significantly different compared with the healthy controls.2.VIP analysis and metabolic pathway analysis revealed the metabolites and metabolic pathways which significantly differ between SCLC and control groups in men and women.Both of them are similar to the cellular metabolism revealed in vitro cells,and have five same distrubed metabolic pathways including phospholipid metabolic pathway,amino acid metabolic pathway,urea cycle,purine metabolic pathway and fatty acid oxidation.3.Five metabolites from these pathways were selected as potential diagnostic markers and analyzed by ROC curve and 2 × 2 contingency table analysis.The accuracy of the selected marker combinations in distinguishing SCLC from healthy individuals were both more than 97% in men and women.Sensitivity and specificity were above 0.95 in men,while in women,the sensitivity and specificity reached 1.The metabolites combinations of women are different from men,but they are from the same metabolic pathways.Conclusion: 1.The metabolism of SCLC cells and patients with SCLC was significantly different compared with the control groups.2.Comparing the metabolism results of SCLC cells and blood of patients,the changes of some specific metabolites are different from those of the control groups.However,the metabolic pathways of these metabolites are highly similar.They have the same five disturbed metabolic pathways including amino acids,urea cycle,purine,phospholipid metabolic pathway,and fatty acids oxidation metabolic pathway.3.The potential driving genes of SCLC: APRT,CPS1,XDH,HPRT1 and CPT1 A were screened from the cell metabolism of SCLC,and were verified at the cellur level.4.We found a group of potential metabolic tumor markers for SCLC in both male and female patients,which can accurately distinguish SCLC from healthy controls.It may contribute to the early diagnosis and screening of small cell lung cancer.