#METABOLOMICS WORKBENCH Lhoang_2004_20250417_083921 DATATRACK_ID:5848 STUDY_ID:ST003870 ANALYSIS_ID:AN006359 PROJECT_ID:PR002426 VERSION 1 CREATED_ON April 18, 2025, 9:13 am #PROJECT PR:PROJECT_TITLE lipidomics Studies PR:PROJECT_TYPE MS qualitative analysis PR:PROJECT_SUMMARY Lipids are essential for tumours because of their structural, energetic, and PR:PROJECT_SUMMARY signaling roles. While many cancer cells upregulate lipid synthesis, growing PR:PROJECT_SUMMARY evidence suggests that tumours simultaneously intensify the uptake of PR:PROJECT_SUMMARY circulating lipids carried by lipoproteins. Which mechanisms promote the uptake PR:PROJECT_SUMMARY of extracellular lipids, and how this pool of lipids contributes to cancer PR:PROJECT_SUMMARY progression, are poorly understood. Here, using functional genetic screens, we PR:PROJECT_SUMMARY find that lipoprotein uptake confers resistance to lipid peroxidation and PR:PROJECT_SUMMARY ferroptotic cell death. Lipoprotein supplementation robustly inhibits PR:PROJECT_SUMMARY ferroptosis across numerous cancer types, an effect largely driven by PR:PROJECT_SUMMARY lipoprotein delivery of α-tocopherol, the most abundant form of vitamin E. PR:PROJECT_SUMMARY Mechanistically, cancer cells take up lipoproteins through a pathway dependent PR:PROJECT_SUMMARY on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. PR:PROJECT_SUMMARY Tumour GAGs are a major determinant of the uptake of both low and high density PR:PROJECT_SUMMARY lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of PR:PROJECT_SUMMARY surface GAGs decreases the uptake of lipoproteins, sensitizes cells to PR:PROJECT_SUMMARY ferroptosis and reduces tumour growth in mice. We also find that human clear PR:PROJECT_SUMMARY cell renal cell carcinomas, a distinctively lipid-rich tumour type, display PR:PROJECT_SUMMARY elevated levels of the GAG chondroitin sulfate and lipoprotein-derived PR:PROJECT_SUMMARY antioxidants compared to non-malignant human kidney. Altogether, our work PR:PROJECT_SUMMARY identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for PR:PROJECT_SUMMARY cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG PR:PROJECT_SUMMARY biosynthesis as a critical mediator of this process. PR:INSTITUTE Scripps Research Institute PR:LAST_NAME Hoang PR:FIRST_NAME Linh PR:ADDRESS 10650 North Torrey Pines Road PR:EMAIL lhoang@scripps.edu PR:PHONE 8587849415 #STUDY ST:STUDY_TITLE Lipidomics studies on B16 cells ST:STUDY_TYPE Lipidomics ST:STUDY_SUMMARY Lipids are essential for tumours because of their structural, energetic, and ST:STUDY_SUMMARY signaling roles. While many cancer cells upregulate lipid synthesis, growing ST:STUDY_SUMMARY evidence suggests that tumours simultaneously intensify the uptake of ST:STUDY_SUMMARY circulating lipids carried by lipoproteins. Which mechanisms promote the uptake ST:STUDY_SUMMARY of extracellular lipids, and how this pool of lipids contributes to cancer ST:STUDY_SUMMARY progression, are poorly understood. Here, using functional genetic screens, we ST:STUDY_SUMMARY find that lipoprotein uptake confers resistance to lipid peroxidation and ST:STUDY_SUMMARY ferroptotic cell death. Lipoprotein supplementation robustly inhibits ST:STUDY_SUMMARY ferroptosis across numerous cancer types, an effect largely driven by ST:STUDY_SUMMARY lipoprotein delivery of α-tocopherol, the most abundant form of vitamin E. ST:STUDY_SUMMARY Mechanistically, cancer cells take up lipoproteins through a pathway dependent ST:STUDY_SUMMARY on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. ST:STUDY_SUMMARY Tumour GAGs are a major determinant of the uptake of both low and high density ST:STUDY_SUMMARY lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of ST:STUDY_SUMMARY surface GAGs decreases the uptake of lipoproteins, sensitizes cells to ST:STUDY_SUMMARY ferroptosis and reduces tumour growth in mice. We also find that human clear ST:STUDY_SUMMARY cell renal cell carcinomas, a distinctively lipid-rich tumour type, display ST:STUDY_SUMMARY elevated levels of the GAG chondroitin sulfate and lipoprotein-derived ST:STUDY_SUMMARY antioxidants compared to non-malignant human kidney. Altogether, our work ST:STUDY_SUMMARY identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for ST:STUDY_SUMMARY cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG ST:STUDY_SUMMARY biosynthesis as a critical mediator of this process. ST:INSTITUTE Scripps Research Institute ST:LAST_NAME Hoang ST:FIRST_NAME Linh ST:ADDRESS 10650 North Torrey Pines Road ST:EMAIL lhoang@scripps.edu ST:PHONE 18587849415 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - X011325_Lipidomics_1A Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011325_Lipidomics_1A_pos_P1-A-2_01_23041.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_1B Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011325_Lipidomics_1B_pos_P1-A-5_01_23047.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_1C Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011325_Lipidomics_1C_pos_P1-A-8_01_23055.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_1D Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011325_Lipidomics_1D_pos_P1-B-2_01_23061.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_1E Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011325_Lipidomics_1E_pos_P1-B-5_01_23069.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_2A Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_2A_pos_P1-A-3_01_23043.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_2B Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_2B_pos_P1-A-6_01_23049.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_2C Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_2C_pos_P1-A-9_01_23057.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_2D Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_2D_pos_P1-B-3_01_23065.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_2E Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_2E_pos_P1-B-6_01_23071.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_3A Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_3A_pos_P1-A-4_01_23045.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_3B Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_3B_pos_P1-A-7_01_23053.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_3C Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_3C_pos_P1-B-1_01_23059.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_3D Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_3D_pos_P1-B-4_01_23067.mzML SUBJECT_SAMPLE_FACTORS - X011325_Lipidomics_3E Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011325_Lipidomics_3E_pos_P1-B-7_01_23073.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_1A_neg Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011525_Lipidomics_1A_neg_P1-A-2_01_23120.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_1B_neg Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011525_Lipidomics_1B_neg_P1-A-5_01_23126.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_1C_neg Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011525_Lipidomics_1C_neg_P1-A-8_01_23134.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_1D_neg Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011525_Lipidomics_1D_neg_P1-B-2_01_23140.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_1E_neg Genotype:- | Sample source:B16 Cells | Treatment:Control RAW_FILE_NAME(Raw file name)=011525_Lipidomics_1E_neg_P1-B-5_01_23148.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_2A_neg Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_2A_neg_P1-A-3_01_23122.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_2B_neg Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_2B_neg_P1-A-6_01_23128.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_2C_neg Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_2C_neg_P1-A-9_01_23136.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_2D_neg Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_2D_neg_P1-B-3_01_23144.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_2E_neg Genotype:- | Sample source:B16 Cells | Treatment:received LDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_2E_neg_P1-B-6_01_23150.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_3A_neg Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_3A_neg_P1-A-4_01_23124.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_3B_neg Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_3B_neg_P1-A-7_01_23132.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_3C_neg Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_3C_neg_P1-B-1_01_23138.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_3D_neg Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_3D_neg_P1-B-4_01_23146.mzML SUBJECT_SAMPLE_FACTORS - X011525_Lipidomics_3E_neg Genotype:- | Sample source:B16 Cells | Treatment:received HDL (50ug/ml) RAW_FILE_NAME(Raw file name)=011525_Lipidomics_3E_neg_P1-B-7_01_23152.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were collected, washed with cold PBS and flash-frozen in liquid N2 CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY three groups– one receiving purified LDL (50 µg/mL), one receiving purified TR:TREATMENT_SUMMARY HDL (50 µg/mL), and one receiving neither. Each group consisted of five TR:TREATMENT_SUMMARY replicates with 10 million cells per replicate. Cells from each group were TR:TREATMENT_SUMMARY cultured in their respective condition for 36 hours before being collected. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cold IPA:H2O (4:1) was added to the cell pellets. Samples were vortexed, snap SP:SAMPLEPREP_SUMMARY frozen in liquid nitrogen, thawed and sonicated in an ice-cold bath. Freeze-thaw SP:SAMPLEPREP_SUMMARY cycles were repeated two more times before storing samples at -20°C for 1 h. SP:SAMPLEPREP_SUMMARY Samples were centrifuged, collected the supernatant, evaporated and SP:SAMPLEPREP_SUMMARY reconstituted in IPA:ACN (1:1) (volume normalized to protein amount using SP:SAMPLEPREP_SUMMARY PierceTM BCA protein assay kits, Thermo Scientific). Samples were transferred to SP:SAMPLEPREP_SUMMARY glass vials with glass inserts and stored at -80°C until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker Impact II Qtof CH:COLUMN_NAME Waters Acquity UPLC BEH C18 (1.0x100mm, 1.7um) CH:SOLVENT_A 50% Water/30% Acetonitrile/20% Isopropyl alcohol; 10mM Ammonium formate CH:SOLVENT_B 90% Isopropyl alcohol/9% Acetonitrile/1% Water; 10mM Ammonium formate CH:FLOW_GRADIENT 0-1.5 min: 10%B, 1.5-3 min: 10-45%B, 3-4min: 45-52%B, 4-6 min: 52-58%B, 6-8 min: CH:FLOW_GRADIENT 58-66%B, 8-10.50min: 66-70%B, 10.50-13.50min: 70-75% B, 13.50-20min: 75-100%, CH:FLOW_GRADIENT 20-24 min: 100%, 24-25 min: 100-10%B, and posttime for 5 min CH:FLOW_RATE 125 µL/min CH:COLUMN_TEMPERATURE 60°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Impact II MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Samples were randomized and analyzed. Raw LC-MS data was converted to the mzData MS:MS_COMMENTS format using Bruker Compassxportsfx1.2. The mzData files were uploaded to XCMS MS:MS_COMMENTS Online for data processing. XCMS-Metlin and MSDial were used for metabolite MS:MS_COMMENTS identification. MS:MS_RESULTS_FILE ST003870_AN006359_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END