| Background Colorectal cancer(CRC),as the third most common fatal tumor in the world,is a common malignant tumor in the gastrointestinal system.The clinical symptoms of early CRC are not obvious.It is often diagnosed at an advanced stage,with limited curative effect and high cost.The most important prevention and treatment strategies for CRC are early screening,early diagnosis and early treatment.Heterogeneous metabolic disorder is an important characteristic of malignant tumor,which provides a biochemical basis for the early screening of colorectal cancer.Proton nuclear magnetic resonance spectroscopy(1H-NMR)-based metabolomics has been shown to be accurately capable of determining the metabolic characteristics of CRC patients.Recently,we have identified distinct NMR-based fecal,serum and urine metabolic signatures respectively,which were be able to discriminate early stage CRC patients from healthy controls,highlighting the potential utility of NMR-based biofluids metabolomics fingerprinting as noninvasive predictors of earlier diagnosis in CRC patients.However,body fluid metabolism is sensitive to many factors,such as genetic composition,food and environment.Targeted metabolic detection of cancer tissue is the direct method to screen tumor related metabolic biomarkers.Therefore,it is necessary to clarify the metabolic characteristics of different stages and different anatomical locations in CRC,so as to provide an evidence for the optimal metabolic model construction through a combination of the biomarkers in fecal extracts,serum and urine for CRC early detection.In this study,high-resolution 600 MHz 1H NMR technique combined with pattern recognition was performed to investigate the metabolic differences of CRC tissues in different stages and different locations,as compared with their paired adjacent noncancerous tissues.In addition,the importance of metabolic pathways was analyzed.Finally,the temporal and spatial heterogeneity of metabolic perturbation of CRC tissues were determined.This study provides an important basis for further constructing the optimal biofluids metabolic model,verifying the relevant molecular mechanisms,as well as studying the intestinal microflora in CRC screening.Methods 106 pairs of colorectal cancer tissues and their corresponding adjacent noncancerous tissues were collected.600 MHz 1H-NMR CPMG pulse sequence was used to acquire the 1H NMR spectra of the colorectal tissues.The original spectra were corrected for frequency correction,baseline distortion and calibrated to the TSP signal at 0.0 ppm.To reduce the complexity of the NMR data,the spectral range from 9.0 to 0.5ppm was segmented into buckets with equal widths of 0.004 ppm each.The region of 5.5–4.5 ppm was discarded to eliminate imperfect water suppression.Each bucket was internally normalized to the total sum of the spectral integrals prior to pattern recognition analysis,to compensate for differences in sample concentration.Pattern recognition was applied on NMR processed data to acquire the detailed metabolic information.The metabolic differences in different stages and different locations of CRC tissues were determined.Furthermore,significant metabolic pathways were analyzed using Metabo Analyst software,with a pathway impact value of greater than or equal to 0.1 and –log(p)value of no less than 2.Finally,the temporal and spatial heterogeneity of metabolic disorders in CRC tissues were defined.Results 1.Our study revealed a significant number of altered metabolites in CRC tissues as compared with the adjacent noncancerous tissues,including higher levels of leucine,isoleucine,valine,alanine,lactic acid,alanine,glutamic acid,succinic acid,asparagine,phosphocholine,L-carnitine,taurine,glycine,serine,tyrosine,phenylalanine,tryptophan,hypoxanthine and cysteine,together with lower levels of butyric acid,glucose,glutamine,creatine,creatinine,inositol and glutathione.2.There were significant differences in tissue metabolism among different stages of CRC.The levels of butyric acid,propionic acid,acetic acid,leucine,isoleucine,valine,glutamic acid in stage of T3-4 were higher than those in stage of T 1-2 in CRC.3.The differences of tissues metabolism in different locations of CRC were statistically significant.Higher levels of citric acid,glucose,tyrosine,and lower levels of choline phosphate and hypoxanthine were observed in colon cancer tissues,as compared to those in rectal cancer tissues.The amounts of L-carnitine and inositol in left colon cancer tissues were significantly higher,while those of propionic acid,lysine,succinic acid and glucose were markedly decreased in left colon cancer tissues,compared to those in left colon tissues.4.The importance of metabolic pathway analysis showed that taurine and hypotaurine metabolism was the most affected pathway in stage of T1-2 CRC,while alanine,aspartate and glutamate metabolism was the most affected pathway in stage T3-4 CRC.Perturbation of glycine,serine and threonine metabolism was most strongly associated with both colon and rectal cancer progression.Furthermore,disorders of alanine,aspartate and glutamate metabolism was found to be significant in left colon cancer tissues,while the metabolic disorders of glycine,serine and threonine was significant in right colon cancer tissues.Conclusions 1.Compared with adjacent noncancerous tissues,many of the metabolite levels in CRC tissues were significantly altered,indicating deregulation of metabolic pathways,such as glucose metabolism,one-carbon metabolism,glutamine metabolism,amino acid metabolism,fatty acid metabolism,TCA cycle,choline metabolism and redox homeostasis.2.Significant metabolic differences were found in CRC tissues at different pathological stages and different sites,suggesting that the metabolic reprogramming of CRC are temporally and spatially heterogenous. |
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