#METABOLOMICS WORKBENCH MetaBalloon_20250424_122928 DATATRACK_ID:5861 STUDY_ID:ST003898 ANALYSIS_ID:AN006401 PROJECT_ID:PR002439 VERSION 1 CREATED_ON May 6, 2025, 1:58 pm #PROJECT PR:PROJECT_TITLE Measuring α-tocopherol and cholesterol from ccRCC tissues PR:PROJECT_SUMMARY Lipids are essential components of cancer cells due to their structural and PR:PROJECT_SUMMARY signaling roles. To meet metabolic demands, many cancers take up extracellular PR:PROJECT_SUMMARY lipids; however, how these lipids contribute to cancer growth and progression PR:PROJECT_SUMMARY remains poorly understood. Here, using functional genetic screens, we identify PR:PROJECT_SUMMARY lipoprotein uptake—the primary mechanism for lipid transport in PR:PROJECT_SUMMARY circulation—as a key determinant of ferroptosis sensitivity in cancer. PR:PROJECT_SUMMARY Lipoprotein supplementation robustly inhibits ferroptosis across numerous cancer PR:PROJECT_SUMMARY types, an effect largely driven by lipoprotein delivery of α-tocopherol, the PR:PROJECT_SUMMARY most abundant form of vitamin E. Mechanistically, cancer cells take up PR:PROJECT_SUMMARY lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) PR:PROJECT_SUMMARY linked to cell-surface proteoglycans. Disrupting GAG biosynthesis or acutely PR:PROJECT_SUMMARY degrading surface GAGs reduces lipoprotein uptake, sensitizes cancer cells to PR:PROJECT_SUMMARY ferroptosis, and impairs tumour growth in mice. Notably, human clear cell renal PR:PROJECT_SUMMARY cell carcinomas (ccRCC), a lipid-rich malignancy, exhibit elevated levels of PR:PROJECT_SUMMARY chondroitin sulfate and increased lipoprotein-derived α-tocopherol compared to PR:PROJECT_SUMMARY normal kidney tissue. Altogether, our work establishes lipoprotein uptake as a PR:PROJECT_SUMMARY critical anti-ferroptotic mechanism in cancer and implicates GAG biosynthesis as PR:PROJECT_SUMMARY a therapeutic target. PR:INSTITUTE University of Texas Southwestern Medical Center at Dallas PR:DEPARTMENT Children's Research Institute PR:LABORATORY Metabolomics Facility PR:LAST_NAME Cai PR:FIRST_NAME Feng PR:ADDRESS 6000 Harry Hines Blvd. PR:EMAIL feng.cai@utsouthwestern.edu PR:PHONE 2146483056 #STUDY ST:STUDY_TITLE Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ST:STUDY_TITLE ferroptosis ST:STUDY_SUMMARY Lipids are essential components of cancer cells due to their structural and ST:STUDY_SUMMARY signaling roles and many cancers take up extracellular lipids to meet metabolic ST:STUDY_SUMMARY demands. How these lipids contribute to cancer growth and progression remains ST:STUDY_SUMMARY poorly understood. Using functional genetic screens, lipoprotein uptake—the ST:STUDY_SUMMARY primary mechanism for lipid transport in circulation—is identify as a key ST:STUDY_SUMMARY determinant of ferroptosis sensitivity in cancer. Lipoprotein supplementation ST:STUDY_SUMMARY robustly inhibits ferroptosis across numerous cancer types, an effect largely ST:STUDY_SUMMARY driven by lipoprotein delivery of α-tocopherol. Mechanistically, cancer cells ST:STUDY_SUMMARY take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans ST:STUDY_SUMMARY (GAGs) linked to cell-surface proteoglycans. Disrupting GAG biosynthesis or ST:STUDY_SUMMARY acutely degrading surface GAGs reduces lipoprotein uptake, sensitizes cancer ST:STUDY_SUMMARY cells to ferroptosis, and impairs tumor growth in mice. Notably, human clear ST:STUDY_SUMMARY cell renal cell carcinomas (ccRCC), a lipid-rich malignancy, exhibit elevated ST:STUDY_SUMMARY levels of chondroitin sulfate and increased lipoprotein-derived α-tocopherol ST:STUDY_SUMMARY compared to normal kidney tissue. Altogether, this work establishes lipoprotein ST:STUDY_SUMMARY uptake as a critical anti-ferroptotic mechanism in cancer and implicates GAG ST:STUDY_SUMMARY biosynthesis as a therapeutic target. ST:INSTITUTE University of Texas Southwestern Medical Center at Dallas ST:DEPARTMENT Children's Research Institute ST:LABORATORY Metabolomics Facility ST:LAST_NAME Cai ST:FIRST_NAME Feng ST:ADDRESS 6000 Harry Hines Blvd. ST:EMAIL feng.cai@utsouthwestern.edu ST:PHONE 2146483056 #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 - B4 (1) Sample source:Human patient ccRCC tissues | Sample Information:VM91_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=B4 (1).mzML SUBJECT_SAMPLE_FACTORS - B4 (2) Sample source:Human patient ccRCC tissues | Sample Information:VM91_Primary ccRCC RAW_FILE_NAME(Rawdata files)=B4 (2).mzML SUBJECT_SAMPLE_FACTORS - B4 (3) Sample source:Human patient ccRCC tissues | Sample Information:VM92_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=B4 (3).mzML SUBJECT_SAMPLE_FACTORS - B4 (4) Sample source:Human patient ccRCC tissues | Sample Information:VM92_Primary ccRCC RAW_FILE_NAME(Rawdata files)=B4 (4).mzML SUBJECT_SAMPLE_FACTORS - B4 (5) Sample source:Human patient ccRCC tissues | Sample Information:VM94_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=B4 (5).mzML SUBJECT_SAMPLE_FACTORS - B4 (6) Sample source:Human patient ccRCC tissues | Sample Information:VM94_Primary ccRCC RAW_FILE_NAME(Rawdata files)=B4 (6).mzML SUBJECT_SAMPLE_FACTORS - 9456 (1) Sample source:Human patient ccRCC tissues | Sample Information:VM95_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (1).mzML SUBJECT_SAMPLE_FACTORS - 9456 (2) Sample source:Human patient ccRCC tissues | Sample Information:VM95_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (2).mzML SUBJECT_SAMPLE_FACTORS - 9456 (3) Sample source:Human patient ccRCC tissues | Sample Information:VM96_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (3).mzML SUBJECT_SAMPLE_FACTORS - 9456 (4) Sample source:Human patient ccRCC tissues | Sample Information:VM96_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (4).mzML SUBJECT_SAMPLE_FACTORS - 9456 (5) Sample source:Human patient ccRCC tissues | Sample Information:VM97_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (5).mzML SUBJECT_SAMPLE_FACTORS - 9456 (6) Sample source:Human patient ccRCC tissues | Sample Information:VM97_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (6).mzML SUBJECT_SAMPLE_FACTORS - 9456 (7) Sample source:Human patient ccRCC tissues | Sample Information:VM98_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (7).mzML SUBJECT_SAMPLE_FACTORS - 9456 (8) Sample source:Human patient ccRCC tissues | Sample Information:VM98_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (8).mzML SUBJECT_SAMPLE_FACTORS - 9456 (9) Sample source:Human patient ccRCC tissues | Sample Information:VM100_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (9).mzML SUBJECT_SAMPLE_FACTORS - 9456 (10) Sample source:Human patient ccRCC tissues | Sample Information:VM100_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (10).mzML SUBJECT_SAMPLE_FACTORS - 9456 (11) Sample source:Human patient ccRCC tissues | Sample Information:VM101_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (11).mzML SUBJECT_SAMPLE_FACTORS - 9456 (12) Sample source:Human patient ccRCC tissues | Sample Information:VM101_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (12).mzML SUBJECT_SAMPLE_FACTORS - 9456 (13) Sample source:Human patient ccRCC tissues | Sample Information:VM102_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (13).mzML SUBJECT_SAMPLE_FACTORS - 9456 (14) Sample source:Human patient ccRCC tissues | Sample Information:VM102_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (14).mzML SUBJECT_SAMPLE_FACTORS - 9456 (15) Sample source:Human patient ccRCC tissues | Sample Information:VM103_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (15).mzML SUBJECT_SAMPLE_FACTORS - 9456 (16) Sample source:Human patient ccRCC tissues | Sample Information:VM103_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (16).mzML SUBJECT_SAMPLE_FACTORS - 9456 (17) Sample source:Human patient ccRCC tissues | Sample Information:VM104_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (17).mzML SUBJECT_SAMPLE_FACTORS - 9456 (18) Sample source:Human patient ccRCC tissues | Sample Information:VM104_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (18).mzML SUBJECT_SAMPLE_FACTORS - 9456 (19) Sample source:Human patient ccRCC tissues | Sample Information:VM105_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (19).mzML SUBJECT_SAMPLE_FACTORS - 9456 (20) Sample source:Human patient ccRCC tissues | Sample Information:VM105_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (20).mzML SUBJECT_SAMPLE_FACTORS - 9456 (21) Sample source:Human patient ccRCC tissues | Sample Information:VM106_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (21).mzML SUBJECT_SAMPLE_FACTORS - 9456 (22) Sample source:Human patient ccRCC tissues | Sample Information:VM106_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (22).mzML SUBJECT_SAMPLE_FACTORS - 9456 (23) Sample source:Human patient ccRCC tissues | Sample Information:VM107_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (23).mzML SUBJECT_SAMPLE_FACTORS - 9456 (24) Sample source:Human patient ccRCC tissues | Sample Information:VM107_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (24).mzML SUBJECT_SAMPLE_FACTORS - 9456 (25) Sample source:Human patient ccRCC tissues | Sample Information:VM108_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (25).mzML SUBJECT_SAMPLE_FACTORS - 9456 (26) Sample source:Human patient ccRCC tissues | Sample Information:VM108_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (26).mzML SUBJECT_SAMPLE_FACTORS - 9456 (27) Sample source:Human patient ccRCC tissues | Sample Information:VM109_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (27).mzML SUBJECT_SAMPLE_FACTORS - 9456 (28) Sample source:Human patient ccRCC tissues | Sample Information:VM109_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (28).mzML SUBJECT_SAMPLE_FACTORS - 9456 (29) Sample source:Human patient ccRCC tissues | Sample Information:VM110_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (29).mzML SUBJECT_SAMPLE_FACTORS - 9456 (30) Sample source:Human patient ccRCC tissues | Sample Information:VM110_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (30).mzML SUBJECT_SAMPLE_FACTORS - 9456 (31) Sample source:Human patient ccRCC tissues | Sample Information:VM111_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (31).mzML SUBJECT_SAMPLE_FACTORS - 9456 (32) Sample source:Human patient ccRCC tissues | Sample Information:VM111_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (32).mzML SUBJECT_SAMPLE_FACTORS - 9456 (33) Sample source:Human patient ccRCC tissues | Sample Information:VM112_Adjacent Kidney RAW_FILE_NAME(Rawdata files)=9456 (33).mzML SUBJECT_SAMPLE_FACTORS - 9456 (34) Sample source:Human patient ccRCC tissues | Sample Information:VM112_Primary ccRCC RAW_FILE_NAME(Rawdata files)=9456 (34).mzML #COLLECTION CO:COLLECTION_SUMMARY We collected 50-100 mg of human ccRCC or adjacent kidney tissues, resuspended CO:COLLECTION_SUMMARY them in 800 µl of PBS, lysed using a BeadBlaster 24R (Benchmark Scientific) CO:COLLECTION_SUMMARY followed by sample sonication for 60 seconds. 1/10 of this solution was CO:COLLECTION_SUMMARY collected for protein quantification for normalization of values. The remaining CO:COLLECTION_SUMMARY supernatant was processed as following: addition of 700 µL of LC/MS grade CO:COLLECTION_SUMMARY ethanol (EtOH) + 2.1 mL of LC/MS grade hexane (Sigma). Solutions were then CO:COLLECTION_SUMMARY thoroughly vortexed for 5 minutes at 4°C. After centrifugation, the upper layer CO:COLLECTION_SUMMARY was collected into a new tube. Next, we re-extracted the remaining aqueous phase CO:COLLECTION_SUMMARY by adding 300 µL of EtOH + 900 µL of hexane, followed by vortexing and CO:COLLECTION_SUMMARY centrifugation. The two non-polar phases containing vitamin E and cholesterol CO:COLLECTION_SUMMARY were then collected together, dried down and stored at -70°C until analysis. CO:SAMPLE_TYPE Tumor tissue, Adjacent kidney tissue #TREATMENT TR:TREATMENT_SUMMARY no treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Pellets are reconstituted into LC/MS grade ethanol (EtOH, 200 µl) and SP:SAMPLEPREP_SUMMARY centrifuged at 21,000 G for 15 min, the supernatants are transferred into LC-MS SP:SAMPLEPREP_SUMMARY vials for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu 20AD CH:COLUMN_NAME Phenomenex Synergi Polar-RP (150 x 2mm, 4um, 80Å) CH:SOLVENT_A 65% methanol/35% water; 2.0 mM ammonium acetate CH:SOLVENT_B 63% methanol/37% isopropanol; 2.0 mM ammonium acetate CH:FLOW_GRADIENT 0–2.0 min, linear gradient 0–60% B, 2.0–5.6 min, linear gradient 60–100% CH:FLOW_GRADIENT B, then the column was washed with 100% B for 4.4 min before reconditioning it CH:FLOW_GRADIENT for 5 min using 0% B CH:FLOW_RATE 0.5mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE APCI MS:ION_MODE POSITIVE MS:MS_COMMENTS An AB SCIEX 5500 QTRAP liquid chromatography/mass spectrometer (Applied MS:MS_COMMENTS Biosystems SCIEX), equipped with a vacuum degasser, a quaternary pump, an MS:MS_COMMENTS autosampler, a thermostatted column compartment, and a triple MS:MS_COMMENTS quadrupole/ion-trap mass spectrometer with atmospheric pressure chemical MS:MS_COMMENTS ionization interface, controlled by AB SCIEX Analyst 1.6.1 Software. The MRMs MS:MS_COMMENTS (Q1/Q3) used for metabolites were 369.0/161.0 (cholesterol, CE: 15), 431/165 MS:MS_COMMENTS (α-tocopherol, CE: 30), and 437/171 (α-tocopherol-d6, internal standard, CE: MS:MS_COMMENTS 30). The Multiquant 3.0 software was used to identify peak and integration. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion count MS_METABOLITE_DATA_START Samples B4 (1) B4 (2) B4 (3) B4 (4) B4 (5) B4 (6) 9456 (1) 9456 (2) 9456 (3) 9456 (4) 9456 (5) 9456 (6) 9456 (7) 9456 (8) 9456 (9) 9456 (10) 9456 (11) 9456 (12) 9456 (13) 9456 (14) 9456 (15) 9456 (16) 9456 (17) 9456 (18) 9456 (19) 9456 (20) 9456 (21) 9456 (22) 9456 (23) 9456 (24) 9456 (25) 9456 (26) 9456 (27) 9456 (28) 9456 (29) 9456 (30) 9456 (31) 9456 (32) 9456 (33) 9456 (34) Factors Sample source:Human patient ccRCC tissues | Sample Information:VM91_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM91_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM92_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM92_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM94_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM94_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM95_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM95_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM96_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM96_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM97_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM97_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM98_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM98_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM100_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM100_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM101_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM101_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM102_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM102_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM103_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM103_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM104_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM104_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM105_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM105_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM106_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM106_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM107_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM107_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM108_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM108_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM109_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM109_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM110_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM110_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM111_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM111_Primary ccRCC Sample source:Human patient ccRCC tissues | Sample Information:VM112_Adjacent Kidney Sample source:Human patient ccRCC tissues | Sample Information:VM112_Primary ccRCC cholesterol 49757041.87 26099240.39 42550751.38 47908626.35 35376459.57 45176628.55 1208397.657 10157639.96 22255444.79 8727356.865 4927180.894 8840102.263 2499124.279 6722116.611 2642728.428 15194656.88 15016464.06 24403153.2 2626223.966 21073730.48 6492292.001 739789.0528 2161950.272 4476265.832 21413701.04 1329352.006 14016510.73 34791.2484 24254783.88 33825985.89 7121480.96 6258576.074 20436654.85 53317832.83 8514631.636 52279881.13 6797520.984 15918860.63 4206069.238 18293914.82 Vitamin E (α-tocopherol) 341067.9308 2279444.488 286014.6393 43094126.46 4776147.533 10296634.73 1789.923432 743943.7224 673220.2051 3947188.666 58366.08008 3344374.812 63420.04656 238303.53 1 2556509.465 640079.0257 1972628.373 28747.22087 1909302.637 81171.50419 317023.3955 200156.1211 665574.5114 221725.0954 40460.45201 203133.7461 1 2256794.705 7130929.599 93381.00085 621686.5748 84352.71079 1199690.952 7098.702395 340742.7639 25483.59417 3851634.299 10018.12997 3734824.906 Vitamin E_d6 (IS) 57881.97862 159879.9683 62567.83537 106619.9665 75922.35632 184942.4005 1148.169728 48035.13249 53883.21646 16485.32836 19143.36536 42125.01377 2456.938311 8064.433156 11381.94729 17865.44024 69910.04398 67201.49968 10079.96627 45522.13707 12250.10209 583.1108315 10980.6951 44939.90818 24864.67857 14187.62451 35543.99085 549.4457726 47640.48745 40816.6111 30335.43964 26213.34801 28191.63853 14758.48584 54783.85673 23601.85609 14678.06046 42687.81959 12470.51637 99458.19392 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID cholesterol C00187 Vitamin E (α-tocopherol) C02477 Vitamin E_d6 (IS) - METABOLITES_END #END