#METABOLOMICS WORKBENCH Linyan_20231216_014858 DATATRACK_ID:4528 STUDY_ID:ST003015 ANALYSIS_ID:AN004948 PROJECT_ID:PR001876 VERSION 1 CREATED_ON December 18, 2023, 5:42 pm #PROJECT PR:PROJECT_TITLE NMR- and MS-based omics reveal characteristic metabolome atlas and optimize PR:PROJECT_TITLE biofluid earlydiagnostic biomarkers for esophageal squamous cell carcinoma PR:PROJECT_SUMMARY Metabolic changes precede malignant histology. However, it remains unclear PR:PROJECT_SUMMARY whether detectable characteristic metabolome exists in esophageal squamous cell PR:PROJECT_SUMMARY carcinoma (ESCC) tissues and biofluids for early diagnosis. We conducted NMR- PR:PROJECT_SUMMARY and MS-based metabolomics on 1,153 matched ESCC tissues, normal mucosae, pre- PR:PROJECT_SUMMARY and one-week post-operative sera and urines from 560 participants across three PR:PROJECT_SUMMARY hospitals, with machine learning, logistic regression and WGCNA. Aberrations in PR:PROJECT_SUMMARY 'alanine, aspartate and glutamate metabolism' proved to be prevalent throughout PR:PROJECT_SUMMARY the ESCC evolution, and were reflected in 16 serum and 10 urine metabolic PR:PROJECT_SUMMARY signatures that were consistently identified by NMR and MS in both discovery and PR:PROJECT_SUMMARY validation sets. NMR-based simplified panels of any five serum or urine PR:PROJECT_SUMMARY metabolites outperformed clinical serological tumor markers (AUC = 0.984 and PR:PROJECT_SUMMARY 0.930, respectively), and were effective in distinguishing early-stage ESCC in PR:PROJECT_SUMMARY test set (serum accuracy = 0.994, urine accuracy = 0.879). Collectively, PR:PROJECT_SUMMARY NMR-based biofluid screening can reveal characteristic metabolic events of ESCC PR:PROJECT_SUMMARY and be feasible for early detection (ChiCTR2300073613). PR:INSTITUTE Radiology Department, Second Affiliated Hospital, Shantou University Medical PR:INSTITUTE College, Shantou PR:LAST_NAME Lin PR:FIRST_NAME Yan PR:ADDRESS No. 69, Dongxia North Road, Shantou, Guangdong, China PR:EMAIL 994809889@qq.com PR:PHONE +86 18823992148 #STUDY ST:STUDY_TITLE NMR- and MS-based omics reveal characteristic metabolome atlas and optimize ST:STUDY_TITLE biofluid earlydiagnostic biomarkers for esophageal squamous cell carcinoma ST:STUDY_TITLE (part-Ⅰ) ST:STUDY_SUMMARY Metabolic changes precede malignant histology. However, it remains unclear ST:STUDY_SUMMARY whether detectable characteristic metabolome exists in esophageal squamous cell ST:STUDY_SUMMARY carcinoma (ESCC) tissues and biofluids for early diagnosis. We conducted NMR- ST:STUDY_SUMMARY and MS-based metabolomics on 1,153 matched ESCC tissues, normal mucosae, pre- ST:STUDY_SUMMARY and one-week post-operative sera and urines from 560 participants across three ST:STUDY_SUMMARY hospitals, with machine learning, logistic regression and WGCNA. Aberrations in ST:STUDY_SUMMARY 'alanine, aspartate and glutamate metabolism' proved to be prevalent throughout ST:STUDY_SUMMARY the ESCC evolution, and were reflected in 16 serum and 10 urine metabolic ST:STUDY_SUMMARY signatures that were consistently identified by NMR and MS in both discovery and ST:STUDY_SUMMARY validation sets. NMR-based simplified panels of any five serum or urine ST:STUDY_SUMMARY metabolites outperformed clinical serological tumor markers (AUC = 0.984 and ST:STUDY_SUMMARY 0.930, respectively), and were effective in distinguishing early-stage ESCC in ST:STUDY_SUMMARY test set (serum accuracy = 0.994, urine accuracy = 0.879). Collectively, ST:STUDY_SUMMARY NMR-based biofluid screening can reveal characteristic metabolic events of ESCC ST:STUDY_SUMMARY and be feasible for early detection (ChiCTR2300073613). ST:INSTITUTE Radiology Department, Second Affiliated Hospital, Shantou University Medical ST:INSTITUTE College, Shantou ST:LAST_NAME Lin ST:FIRST_NAME Yan ST:ADDRESS No. 69, Dongxia North Road, Shantou, Guangdong, China ST:EMAIL 994809889@qq.com ST:PHONE +86 18823992148 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 20220725.1.fid#1 Fator:Tumor tissue RAW_FILE_NAME=Raw_ESCC Tissue.1.fid SUBJECT_SAMPLE_FACTORS - 20220725.2.fid#2 Fator:Tumor tissue RAW_FILE_NAME=Raw_ESCC Tissue.2.fid SUBJECT_SAMPLE_FACTORS - 20220725.3.fid#3 Fator:Tumor tissue RAW_FILE_NAME=Raw_ESCC Tissue.3.fid SUBJECT_SAMPLE_FACTORS - 20220725.4.fid#4 Fator:Tumor tissue RAW_FILE_NAME=Raw_ESCC Tissue.4.fid SUBJECT_SAMPLE_FACTORS - 20220725.5.fid#5 Fator:Tumor tissue RAW_FILE_NAME=Raw_ESCC Tissue.5.fid SUBJECT_SAMPLE_FACTORS - 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20220725.97.fid#97 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.97.fid SUBJECT_SAMPLE_FACTORS - 20220725.98.fid#98 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.98.fid SUBJECT_SAMPLE_FACTORS - 20220725.99.fid#99 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.99.fid SUBJECT_SAMPLE_FACTORS - 20220725.100.fid#100 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.100.fid SUBJECT_SAMPLE_FACTORS - 20220725.101.fid#101 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.101.fid SUBJECT_SAMPLE_FACTORS - 20220725.102.fid#102 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.102.fid SUBJECT_SAMPLE_FACTORS - 20220725.103.fid#103 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.103.fid SUBJECT_SAMPLE_FACTORS - 20220725.104.fid#104 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.104.fid SUBJECT_SAMPLE_FACTORS - 20220725.105.fid#105 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.105.fid SUBJECT_SAMPLE_FACTORS - 20220725.106.fid#106 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.106.fid SUBJECT_SAMPLE_FACTORS - 20220725.107.fid#107 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.107.fid SUBJECT_SAMPLE_FACTORS - 20220725.108.fid#108 Fator:Normal tissue RAW_FILE_NAME=Raw_ESCC Tissue.108.fid #COLLECTION CO:COLLECTION_SUMMARY Tissue samples, including tumor and normal areas 5 cm away, were obtained under CO:COLLECTION_SUMMARY the guidance of experienced pathologists without compromising the patients' CO:COLLECTION_SUMMARY pathology examinations. The collected tissue was rinsed with PBS to avoid CO:COLLECTION_SUMMARY contamination, excess moisture was removed, and it was rapidly frozen in liquid CO:COLLECTION_SUMMARY nitrogen to arrest enzymatic or chemical reactions. Samples were stored at CO:COLLECTION_SUMMARY −80°C until metabolite extraction. CO:COLLECTION_PROTOCOL_FILENAME Tissue_collection_uploaded.pdf CO:SAMPLE_TYPE Tissue CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissue homogenate preparation: Tissue samples weighing 300 mg were ground using SP:SAMPLEPREP_SUMMARY a 60 Hz grinder at 4 °C for 1 min in a mixture of 4 mL/g of CH3OH and 2 mL/g of SP:SAMPLEPREP_SUMMARY ultrapure water. The resulting homogenate was then subjected to vortexing for 1 SP:SAMPLEPREP_SUMMARY min after adding 4 mL/g of CHCl3 and 4 mL/g of ultrapure water. The mixture was SP:SAMPLEPREP_SUMMARY allowed to settle on ice for 15 mins and subsequently centrifuged at 10,000 rpm SP:SAMPLEPREP_SUMMARY for 10 mins at 4 °C. The supernatant was carefully transferred to a new 5 mL SP:SAMPLEPREP_SUMMARY Eppendorf (EP) tube and treated with running nitrogen to remove the methanol. SP:SAMPLEPREP_SUMMARY The resulting liquid was freeze-dried at −80°C until further analysis. The SP:SAMPLEPREP_SUMMARY freeze-dried powder was dissolved in 550 μL of PBS/D2O buffer (pH 7.4, 150 mM), SP:SAMPLEPREP_SUMMARY which contained 0.05% TMSP-2,2,3,3-D4 (D, 98%) SODIUM-3-TRIMETHYLSILYLPROPIONATE SP:SAMPLEPREP_SUMMARY (TSP, Cambridge Isotope Laboratories (CIL), Inc. #DLM-48, CAS #24493-21-8)). SP:SAMPLEPREP_SUMMARY After thorough mixing, the solution was centrifuged at 10,000 rpm for 10 min at SP:SAMPLEPREP_SUMMARY 4 °C. Finally, 500 μL of the supernatant was transferred into a 5 mm NMR tube SP:SAMPLEPREP_SUMMARY (NORELL, #S55 SECURE SERIES) for analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME Tissue_preparation.pdf SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE 4℃ #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME 600 MHz Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_SOLVENT H2O+D2O NM:NMR_RESULTS_FILE ST003015_AN004948_Results.txt UNITS:gauss #END