Metabolomic Analysis Of Breast Cancer Cells Cultured Under Hypoxia Based On GC/MS And LC/MS

The interaction between cancer cells and its microenvironments is one of the hallmarks of cancer metabolic reprogramming.Hypoxia is an important factor of various microenvironments that influence cancer metabolism.Studies have found that hypoxia commonly occurs in solid tumors and acts as a critical microenvironmental factor in the metabolism regulation,invasion and metastasis.There are plenty of existing results based on genomics and transcriptomics researches about hypoxia-related metabolic regulatory mechanism.HIF,induced under hypoxia condition,can regulate multiple metabolism-related genes and enzymes,which inturn affect the tumor metabolism.However,the simple inhibition of HIF-mediated pathways could not always slow down cancer growth,suggesting the existence of HIF-independent regulation mechanisms for hypoxia responses.Studies have reported some genes regulated by hypoxia but independent of HIF.Specially,some significantly changed metabolites under hypoxia would affect the transmission of signaling pathways and further involved in the hypoxia response.Thus the discovery of oncometabolites provides a novel approach for the exploration of hypoxia response mechanism.Cell metabolomics,especially the stable isotope assisted metabolomics,has been more and more widely used in the researches of cancer metabolism for its advantage in the analysis of metabolites and metabolic pathways.Most of the published researches on cancer hypoxia hare based on the results of cellular and molecular biology,with some downstream verification studies using metabolomics techniques targeted to specific metabolites or pathways,while few studies has been reported on the whole metabolome responses to hypoxia using non-targeted metabolomics approaches.Many studies have demonstrated that metabolites can be involved in signal transduction and epigenetic regulation,inducing carsinogenesis.Consequently,integrated metabolomic profiling of cancer cells in specific condition can thus be used as an exploratory method for the screening of oncometabolites,providing new clues for the biology research.Further experiments using stable isotope assisted metabolomics can be utilized to analyze the changes of specific metabolites and metabolic pathways,uncover novel hypoxia-related regulatory mechanism,providing new evidences for the studies on carsinogenesis.In this research,we performed GC/MS and LC/MS based metabolomics analysis to invesitigate the influence of hypoxia on the metabolism of breast cancer cells through the discovering of differential metabolites and metabolic pathways,exploring the hypoxia-related metabolic regulation mechanism.Further,stable isotope assisted metabolomic techniques based on GC/MS and LC/MS were established to ananlyse the specific changes in central carbon metabolism and anaplerotic metabolic pathway based on the results of untargeted metabolomics,to achieve a comprehensive analysis of hypoxia-related influence on breast cancer cells.My researches consist of four parts:In the first part,we established a stable and reliable GC/MS based metabolomic method,analysed the intracellular metabolites of triple negative breast cancer cell line MDA-MB-231,ER⁺breast cancer cell line MCF7 and normal breast epithelial cell line MCF10a cultured under normoxia and hypoxia.Multivariate statistical analysis revealed 33,26 and 17 significantly changed metabolites respectively in these cell lines,among which there were 6 common metabolites mainly belonging to amino acids.In hypoxia,23 metabolites were decreased in MDA-MB-231,which include glucose,intermediates of PPP,amino acids?excluding serine?and lipids,and 10 metabolites were increased,including intermediates of glycolysis and nucleotide metabolism,serine,2-hydroxyglutarate,phosphorylethanolamine and putrescine.In MCF7,hypoxia resulted in the significant decrease of 21 metabolites including glucose,intermediates of TCA,amino acids and lipids,and significant increase of 5 metabolites including intermediates of glycolysis and nucleotide metabolism and putrescine in MCF7.Seventeen metabolites of MCF10a were decreased in hypoxia,including lactate,citrate,amino acids and lipids.Hypoxia resulted in increased glycolysis and decreased TCA cycle in MDA-MB-231 and MCF7.No significantly changed metabolites in PPP were detected in MCF7 and MCF10a under hypoxia;the utilization of glucose in MCF10a in hypoxia were moderately decreased.In addition,ornithine and polyamine were also detected with no statistical significance.Compared with MCF10a,MDA-MB-231 and MCF7 display typical HIF-1a regulation related metabolic changes.All above metabolic changes might be related to the adaptation to hypoxia and growth of cancer cells.In the second part,we performed LC/MS based metabolomics for MDA-MB-231cultured under normoxia and hypoxia.Similar with the results from GC/MS analysis,in hypoxia 6-P-glucose,the intermediates of glycolysis,was increased,whereas TCA cycle intermediates and some amino acids were decreased.Compared with the first part,most of the metabolites detected using LC/MS are polar and of larger molecular weight.In addition to the metabolites of glycolysis and TCA cycle as well as some amino acids,we also detected nucleotides?UMP,dCDP,XMP,GMP and ATP?and its derivates such as NAD⁺,NADP⁺,CoA and fatty acids.Thus LC/MS based metabolomic analysis are necessary complement to GC/MS based metabolomics.In the third part,we establish a stable isotope assisted metabolomics based on GC/MS to explore changes in glucose metabolism and glutamine anaplerotic metabolic pathway based on the results of untargeted metabolomics,to achieve a more comprehensive analysis for the research of hypoxia-related influence on breast cancer cells.With ¹³C-glucose as a substrate in media,we analysed glucose metabolism and found that TCA cycle in MDA-MB-231 and MCF7 was inhibited in hypoxia,which was consistent with the result of the untargeted metabolomics.In addition,there was an increase in pyruvate derived from PPP in MDA-MB-231 in hypoxia,whereas no such changes were detected in MCF7.On the other hand,in the untargeted metabolomic analysis,the intermediates of PPP were decreased in MDA-MB-231 under hypoxia and also no significant changes were detected in MCF7.In hypoxia,the unlabeled ratio of TCA cycle intermediates was increased in MDA-MB-231 and MCF7,indicating the increased metabolism from pathways other than glucose.By using ¹³C-glutamateas substrate,we found upregulated glutamine anaplerotic metabolism and reductive carboxylation.In addition,the conversion from malate to pyruvate was increased,indicating changes of malic enzyme expression.The combined utilization of untargeted and stable isotope assisted metabolomics has demonstrated the advantage of stable isotope assisted metabolomics in analyzing certain metabolites and pathways.In the last part,we established a stable isotope assisted metabolomics based on LC/MS.Two types of mass spectrometry were used for data acquisition and the labled metabolites were identified by using two different methods.Eighteen isotope-labeled metabolites were identified using manual searching,including metabolites of glycolysis and TCA cycle and nucleotides.A downregulated TCA cycle and pyruvate carboxylation were found according to a-ketoglutarate/glutamate/aspartate/citrate.With the use of software based automatic identification method,fifty three and thirty six isotope-labeled metabolites were identified in positive and negative ion mode respectively,among which twelve metabolites were significantly changed,including intermediates of glycolysis,nucleotidesand lipids.Manual identification of isotope-labeled metabolites is time-consuming with heavy workload,and can only be targeted to some kind of metabolites,which is easy to ignore potential differential metabolites with special biological significances.Metabolites identified with the automatic method were mainly lipids and nucleotides,which have complex formula and complicated anabolism.Therefore it is difficult to assign the identified metabolites to correct pathways.Effective combination of manual and automatic screening method of isotope-labeled metabolites should be considered for comprehensive detection of the significantly changed metabolites.