#METABOLOMICS WORKBENCH wnsl2820_20250715_211151 DATATRACK_ID:6174 STUDY_ID:ST004067 ANALYSIS_ID:AN006731 PROJECT_ID:PR002553 VERSION 1 CREATED_ON July 23, 2025, 9:50 am #PROJECT PR:PROJECT_TITLE Development of Novel Pancreatic Cancer Diagnostic Biomarkers via Lipidomic PR:PROJECT_TITLE Profiling PR:PROJECT_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy due to late PR:PROJECT_SUMMARY diagnosis and the lack of sensitive screening tools. We recently discovered that PR:PROJECT_SUMMARY tumors depend on circulating fatty acids as a major energy source. Accordingly, PR:PROJECT_SUMMARY we conducted a study to identify screening-diagnostic biomarkers for PDAC in the PR:PROJECT_SUMMARY lipid fraction of patient plasma. In this study, we performed non-targeted PR:PROJECT_SUMMARY lipidomics on human and mouse plasma samples and identified eight concordant PR:PROJECT_SUMMARY lipid metabolites. We then established four targeted-lipidomics PR:PROJECT_SUMMARY platforms—acylcarnitines, phospholipids, fatty acid amides, and PR:PROJECT_SUMMARY sphingolipids—and carried out quantitative analysis in two independent patient PR:PROJECT_SUMMARY cohorts. A combined tertiary analysis of both cohorts revealed 20 lipid species PR:PROJECT_SUMMARY (1 acylcarnitine, 1 sphingolipid, and 18 phospholipids) that achieved an AUC ≥ PR:PROJECT_SUMMARY 0.75 for discriminating PDAC patients from healthy controls. A logistic PR:PROJECT_SUMMARY regression model incorporating 11 or more phospholipids yielded an AUC of PR:PROJECT_SUMMARY 0.9207, which further increased to 0.9427 in the validation cohort upon addition PR:PROJECT_SUMMARY of CA19-9. While extensive validation in larger, multicenter cohorts is PR:PROJECT_SUMMARY necessary, these findings highlight the promise of a lipid biomarker panel and PR:PROJECT_SUMMARY multivariate algorithms as novel metabolic indicators for early PDAC diagnosis. PR:INSTITUTE National Cancer Center Graduate School of Cancer Science and Policy PR:LAST_NAME Kang PR:FIRST_NAME Joon Hee PR:ADDRESS 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 10408, South Korea PR:EMAIL wnsl2820@gmail.com PR:PHONE 82-031-920-2227 #STUDY ST:STUDY_TITLE Development of Novel Pancreatic Cancer Diagnostic Biomarkers via Lipidomic ST:STUDY_TITLE Profiling - Untargeted metabolomics in mice plasma ST:STUDY_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy due to late ST:STUDY_SUMMARY diagnosis and the lack of sensitive screening tools. We recently discovered that ST:STUDY_SUMMARY tumors depend on circulating fatty acids as a major energy source. Accordingly, ST:STUDY_SUMMARY we conducted a study to identify screening-diagnostic biomarkers for PDAC in the ST:STUDY_SUMMARY lipid fraction of patient plasma. In this study, we performed non-targeted ST:STUDY_SUMMARY lipidomics on human and mouse plasma samples and identified eight concordant ST:STUDY_SUMMARY lipid metabolites. We then established four targeted-lipidomics ST:STUDY_SUMMARY platforms—acylcarnitines, phospholipids, fatty acid amides, and ST:STUDY_SUMMARY sphingolipids—and carried out quantitative analysis in two independent patient ST:STUDY_SUMMARY cohorts. A combined tertiary analysis of both cohorts revealed 20 lipid species ST:STUDY_SUMMARY (1 acylcarnitine, 1 sphingolipid, and 18 phospholipids) that achieved an AUC ≥ ST:STUDY_SUMMARY 0.75 for discriminating PDAC patients from healthy controls. A logistic ST:STUDY_SUMMARY regression model incorporating 11 or more phospholipids yielded an AUC of ST:STUDY_SUMMARY 0.9207, which further increased to 0.9427 in the validation cohort upon addition ST:STUDY_SUMMARY of CA19-9. While extensive validation in larger, multicenter cohorts is ST:STUDY_SUMMARY necessary, these findings highlight the promise of a lipid biomarker panel and ST:STUDY_SUMMARY multivariate algorithms as novel metabolic indicators for early PDAC diagnosis. ST:STUDY_SUMMARY This study aims to find plasma metabolite biomarkers for pancreatic cancer using ST:STUDY_SUMMARY mice plasma: control vs KPC mice. ST:INSTITUTE National Cancer Center Graduate School of Cancer Science and Policy ST:LAST_NAME Kang ST:FIRST_NAME Joon Hee ST:ADDRESS 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 10408, South Korea ST:EMAIL wnsl2820@gmail.com ST:PHONE 82-031-920-2227 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male and female #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 Normal_1 Normal_1 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_1_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_1_neg.raw SUBJECT_SAMPLE_FACTORS Normal_2 Normal_2 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_2_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_2_neg.raw SUBJECT_SAMPLE_FACTORS Normal_3 Normal_3 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_3_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_3_neg.raw SUBJECT_SAMPLE_FACTORS Normal_4 Normal_4 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_4_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_4_neg.raw SUBJECT_SAMPLE_FACTORS Normal_5 Normal_5 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_5_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_5_neg.raw SUBJECT_SAMPLE_FACTORS Normal_6 Normal_6 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_6_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_6_neg.raw SUBJECT_SAMPLE_FACTORS Normal_7 Normal_7 Disease:normal | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=Normal_7_pos.raw; RAW_FILE_NAME(Raw_file_neg)=Normal_7_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12M_1 KPC_1 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_1_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_1_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12F_2 KPC_2 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_2_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_2_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12M_3 KPC_3 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_3_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_3_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12M_4 KPC_4 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_4_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_4_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12M_5 KPC_5 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_5_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_5_neg.raw SUBJECT_SAMPLE_FACTORS KPC_12M_6 KPC_6 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_6_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_6_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_7 KPC_7 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_7_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_7_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_8 KPC_8 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_8_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_8_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16M_9 KPC_9 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_9_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_9_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_10 KPC_10 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_10_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_10_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_11 KPC_11 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_11_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_11_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16M_12 KPC_12 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_12_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_12_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16M_13 KPC_13 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_13_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_13_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_14 KPC_14 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_14_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_14_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16M_15 KPC_15 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_15_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_15_neg.raw SUBJECT_SAMPLE_FACTORS KPC_16F_16 KPC_16 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_16_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_16_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20F_17 KPC_17 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_17_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_17_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20M_18 KPC_18 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_18_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_18_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20M_19 KPC_19 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_19_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_19_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20M_20 KPC_20 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_20_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_20_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20F_21 KPC_21 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_21_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_21_neg.raw SUBJECT_SAMPLE_FACTORS KPC_20F_22 KPC_22 Disease:KPC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=KPC_22_pos.raw; RAW_FILE_NAME(Raw_file_neg)=KPC_22_neg.raw SUBJECT_SAMPLE_FACTORS QC1 QC1 Disease:QC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=QC1_pos.raw; RAW_FILE_NAME(Raw_file_neg)=QC1_neg.raw SUBJECT_SAMPLE_FACTORS QC2 QC2 Disease:QC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=QC2_pos.raw; RAW_FILE_NAME(Raw_file_neg)=QC2_neg.raw SUBJECT_SAMPLE_FACTORS QC3 QC3 Disease:QC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=QC3_pos.raw; RAW_FILE_NAME(Raw_file_neg)=QC3_neg.raw SUBJECT_SAMPLE_FACTORS QC4 QC4 Disease:QC | Sample source:mouse_plasma RAW_FILE_NAME(Raw_file_pos)=QC4_pos.raw; RAW_FILE_NAME(Raw_file_neg)=QC4_neg.raw #COLLECTION CO:COLLECTION_SUMMARY Plasma from both normal and spontaneously occurring pancreatic cancer mice was CO:COLLECTION_SUMMARY collected using heparin-coated syringes. Normal C57BL/6J mice (12 weeks old) and CO:COLLECTION_SUMMARY KPC spontaneous pancreatic cancer mice (12–20 weeks old) were used. At the CO:COLLECTION_SUMMARY study endpoint, blood was drawn via cardiac puncture. Collected blood was CO:COLLECTION_SUMMARY immediately placed on ice. Samples were centrifuged at 2,000 × g for 20 minutes CO:COLLECTION_SUMMARY at 4 °C, and the plasma was stored at –80 °C. CO:SAMPLE_TYPE Blood (plasma) CO:COLLECTION_LOCATION heart CO:COLLECTION_FREQUENCY 1 CO:COLLECTION_DURATION 1 point CO:VOLUMEORAMOUNT_COLLECTED 150-200ul CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS 1.8ml micro-tube CO:STORAGE_VIALS 1.8ml micro-tube CO:COLLECTION_TUBE_TEMP 4°C #TREATMENT TR:TREATMENT_SUMMARY Mouse plasma was stored in a deep freezer until analysis. The mice used for the TR:TREATMENT_SUMMARY study did not receive any drug treatment. TR:TREATMENT none TR:TREATMENT_COMPOUND none TR:TREATMENT_ROUTE none TR:TREATMENT_DOSE none TR:TREATMENT_DOSEVOLUME none TR:TREATMENT_DOSEDURATION none TR:TREATMENT_VEHICLE none TR:ANIMAL_VET_TREATMENTS none TR:ANIMAL_ANESTHESIA none TR:ANIMAL_ACCLIMATION_DURATION none TR:ANIMAL_FASTING none TR:ANIMAL_ENDP_EUTHANASIA CO2 TR:ANIMAL_ENDP_TISSUE_COLL_LIST pancrease TR:ANIMAL_ENDP_TISSUE_PROC_METHOD Formalin fixation TR:ANIMAL_ENDP_CLINICAL_SIGNS C57BL/6J: none, KPC: PDAC #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from human (or mouse) plasma using conventional SP:SAMPLEPREP_SUMMARY liquid-liquid extraction procedures. {Bligh,E.G. and Dyer,W.J. SP:SAMPLEPREP_SUMMARY Can.J.Biochem.Physiol. 1959,37:911}. Briefly, 3-4 volumes of chloroform/methanol SP:SAMPLEPREP_SUMMARY (1/2, v/v) were added to 50 μL of human (or mouse) plasma, and then centrifuged SP:SAMPLEPREP_SUMMARY for 15 min. Additional 100 uL of chloroform or water was added if phase SP:SAMPLEPREP_SUMMARY separation could not be achieved. Nonpolar metabolites containing lipids were SP:SAMPLEPREP_SUMMARY collected from the lower organic phase. The organic solution was dried using SP:SAMPLEPREP_SUMMARY vacuum centrifuge, then stored at -20oC until analysis. The dried matter of SP:SAMPLEPREP_SUMMARY organic with 100 μL of methanol, respectively, prior to LC-MS analysis. All SP:SAMPLEPREP_SUMMARY solvents were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo Fischer SP:SAMPLEPREP_SUMMARY Scientific (Waltham, MA). SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACTION_METHOD liquid-liquid extraction SP:EXTRACT_ENRICHMENT Resuspension after vaccum dry SP:EXTRACT_CLEANUP none SP:EXTRACT_STORAGE -20℃ SP:SAMPLE_RESUSPENSION 50uL methanol SP:SAMPLE_DERIVATIZATION none SP:SAMPLE_SPIKING none #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Column - Part Number:Agilent A3000150X021 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Ultimate3000 CH:COLUMN_NAME Agilent Pursuit 5 C18 (150 x 2.1mm, 5um) CH:SOLVENT_A 100% water; 0.1 % formic acid CH:SOLVENT_B 85% methanol/15% isopropanol; 0.1 % formic acid CH:FLOW_GRADIENT 75 to 99.9 % of B for 2 min, hold at 99.9 % of B for 13 min, 99.9 to 75 % of B CH:FLOW_GRADIENT for 0.5 min, and then hold at 75 % of B for 4.5 min CH:FLOW_RATE 300 ul/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo LTQ XL MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Metabolite features, including neutral mass and retention time values, were MS:MS_COMMENTS determined using Compound Discoverer 3.3. HMDB, KEGG, and LipidMaps were used MS:MS_COMMENTS for DB searches with 10 ppm mass accuracy. mzCloud, LipidBlasts, and in-house DB MS:MS_COMMENTS were used for mass spectral MS/MS searches. MS:MS_RESULTS_FILE ST004067_AN006731_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END