#METABOLOMICS WORKBENCH rjfowle_20241031_114553 DATATRACK_ID:5327 STUDY_ID:ST003570 ANALYSIS_ID:AN005865 PROJECT_ID:PR002203 VERSION 1 CREATED_ON November 12, 2024, 6:18 pm #PROJECT PR:PROJECT_TITLE Dietary fructose enhances tumor growth indirectly via interorgan lipid transfer PR:PROJECT_SUMMARY Fructose consumption has increased considerably over the past five decades, PR:PROJECT_SUMMARY largely due to the widespread use of high-fructose corn syrup (HFCS) as a PR:PROJECT_SUMMARY sweetener. It has been proposed that fructose promotes the growth of some tumors PR:PROJECT_SUMMARY by serving as a direct fuel. Here, we show that fructose supplementation PR:PROJECT_SUMMARY enhances tumor growth in animal models of melanoma, breast cancer, and cervical PR:PROJECT_SUMMARY cancer without causing weight gain or insulin resistance. Interestingly, the PR:PROJECT_SUMMARY cancer cells themselves were unable to use fructose readily as a nutrient PR:PROJECT_SUMMARY because they did not express ketohexokinase-C (KHK-C). Primary hepatocytes did PR:PROJECT_SUMMARY express KHK-C, resulting in fructolysis and the excretion of a variety of lipid PR:PROJECT_SUMMARY species, including lysophosphatidylcholines (LPCs). In co-culture experiments, PR:PROJECT_SUMMARY hepatocyte-derived LPCs were consumed by cancer cells and used to generate PR:PROJECT_SUMMARY phosphatidylcholines (PCs), the major phospholipid of cell membranes. In vivo, PR:PROJECT_SUMMARY HFCS supplementation increased several LPC species by >7-fold in serum. PR:PROJECT_SUMMARY Administration of LPCs to mice was sufficient to increase tumor growth. PR:PROJECT_SUMMARY Pharmacological inhibition of ketohexokinase had no direct effect on cancer PR:PROJECT_SUMMARY cells, but it decreased circulating LPC levels and prevented fructose-mediated PR:PROJECT_SUMMARY tumor growth in vivo. These findings reveal that fructose supplementation PR:PROJECT_SUMMARY increases circulating nutrients such as LPCs, which can enhance tumor growth PR:PROJECT_SUMMARY through a cell non-autonomous mechanism. PR:INSTITUTE Washington University in St. Louis PR:DEPARTMENT Chemistry PR:LABORATORY Gary Patti PR:LAST_NAME Fowle-Grider PR:FIRST_NAME Ronald PR:ADDRESS 6101 Washington Blvd Unit 202, SAINT LOUIS, MO, 63112, USA PR:EMAIL rjfowle@wustl.edu PR:PHONE 309-265-7545 #STUDY ST:STUDY_TITLE Hepatocytes transform fructose into polar metabolites that are selectively ST:STUDY_TITLE utilized by cancer cells ST:STUDY_SUMMARY The goal of this project was to determine if hepatocytes can metabolically ST:STUDY_SUMMARY transform fructose into polar metabolites that CaSki cancer cells can uptake and ST:STUDY_SUMMARY metabolize. The CaSki cells do not metabolize fructose directly to much extent, ST:STUDY_SUMMARY but the liver robustly metabolizes fructose. Therefore, we cultured primary ST:STUDY_SUMMARY hepatocytes from C57BL/6 mice in [U-13C] fructose as the only sugar source for ST:STUDY_SUMMARY 24 hours to form hepatocyte-conditioned media. We then transferred this media to ST:STUDY_SUMMARY CaSki cells for 24 hours to form CaSki-conditioned media. LC-MS profiling of ST:STUDY_SUMMARY polar 13C-labeled metabolites of hepatocyte-conditioned media and ST:STUDY_SUMMARY CaSki-conditioned media revealed that 13C-labeled glucose and 13C-labeled ST:STUDY_SUMMARY aspartate are released from hepatocytes and are up taken by CaSki cells. ST:INSTITUTE Washington University in St. Louis ST:DEPARTMENT Chemistry ST:LABORATORY Gary Patti ST:LAST_NAME Fowle-Grider ST:FIRST_NAME Ronald ST:ADDRESS 5630 Pershing Ave ST:EMAIL rjfowle@wustl.edu ST:PHONE 3092657545 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - CaSki CM C13 Fructose 1 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=CaSki CM C13 Fructose 1.mzXML SUBJECT_SAMPLE_FACTORS - CaSki CM C13 Fructose 2 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=CaSki CM C13 Fructose 1.mzXML SUBJECT_SAMPLE_FACTORS - CaSki CM C13 Fructose 3 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=CaSki CM C13 Fructose 1.mzXML SUBJECT_SAMPLE_FACTORS - Hepatocyte CM C13 Fructose 1 Sample source:culture media | Conditioned media cell type:Hepatocytes RAW_FILE_NAME(Raw file name)=Hepatocyte CM C13 Fructose 1.mzXML SUBJECT_SAMPLE_FACTORS - Hepatocyte CM C13 Fructose 2 Sample source:culture media | Conditioned media cell type:Hepatocytes RAW_FILE_NAME(Raw file name)=Hepatocyte CM C13 Fructose 2.mzXML SUBJECT_SAMPLE_FACTORS - Hepatocyte CM C13 Fructose 3 Sample source:culture media | Conditioned media cell type:Hepatocytes RAW_FILE_NAME(Raw file name)=Hepatocyte CM C13 Fructose 3.mzXML #COLLECTION CO:COLLECTION_SUMMARY 10 mM [U-13C] fructose was dissolved in glucose-free DMEM and 10% dialyzed FBS CO:COLLECTION_SUMMARY with 1% penicillin/streptomycin. The medium was used to culture hepatocytes for CO:COLLECTION_SUMMARY 24 hours, yielding hepatocyte-conditioned medium (CM). 50 uL of this CO:COLLECTION_SUMMARY hepatocyte-conditioned medium was then collected and placed in the -80 degree CO:COLLECTION_SUMMARY celsius freezer for subsequent extraction for LC-MS analysis. This CO:COLLECTION_SUMMARY hepatocyte-conditioned medium was then transferred to CaSki cells for 24 hours, CO:COLLECTION_SUMMARY yielding Caski-conditioned medium (CM).50 uL of this CaSki-conditioned medium CO:COLLECTION_SUMMARY was then collected and placed in the -80 degree celsius freezer for subsequent CO:COLLECTION_SUMMARY extraction for LC-MS analysis. CO:SAMPLE_TYPE Culture Media #TREATMENT TR:TREATMENT_SUMMARY 10 mM [U-13C] fructose was dissolved in glucose-free DMEM and 10% dialyzed FBS TR:TREATMENT_SUMMARY with 1% penicillin/streptomycin. The medium was used to culture hepatocytes for TR:TREATMENT_SUMMARY 24 hours, yielding hepatocyte-conditioned medium (CM). This TR:TREATMENT_SUMMARY hepatocyte-conditioned medium was then transferred to CaSki cells for 24 hours, TR:TREATMENT_SUMMARY yielding Caski-conditioned media (CM). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 90 µL of 2:2:1 methanol:acetonitrile:water (M:A:W) was added to 10 µL of cell SP:SAMPLEPREP_SUMMARY culture media sample. The samples were vortexed and placed at -20 ºC for an SP:SAMPLEPREP_SUMMARY hour. The samples then were centrifuged at 4 ºC for 10 minutes. The supernatant SP:SAMPLEPREP_SUMMARY was removed and analyzed by LC/MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC/MS of polar metabolites was performed by using a SeQuant ZIC-pHILIC column CH:CHROMATOGRAPHY_SUMMARY (EMD Millipore) interfaced with an Agilent 6540 Q-TOF. A 150 × 2.1 mm, 5 μm CH:CHROMATOGRAPHY_SUMMARY SeQuant ZIC-pHILIC column was used with an Agilent 1290 Infinity II LC system, CH:CHROMATOGRAPHY_SUMMARY applying methods established previously 65. Mobile-phase solvents had the CH:CHROMATOGRAPHY_SUMMARY following composition: A = 20 mM ammonium acetate in water:acetonitrile (95:5) CH:CHROMATOGRAPHY_SUMMARY and B = 100% acetonitrile. The following linear gradient was used: 0−0.5 min, CH:CHROMATOGRAPHY_SUMMARY 90% B; 0.5−30 min, 90−30% B; 30−31 min, 30% B. Injection volumes were 2 CH:CHROMATOGRAPHY_SUMMARY μL for all experiments. The column compartment was maintained at 45 °C. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME SeQuant ZIC-pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 95% water/5% acetonitrile; 20 mM ammonium acetate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0−0.5 min, 90% B; 0.5−30 min, 90−30% B linear gradient; 30−31 min, 30% B CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6540 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS LC/MS of polar metabolites was performed by using a SeQuant ZIC-pHILIC column MS:MS_COMMENTS (EMD Millipore) interfaced with an Agilent 6540 Q-TOF. The mass range was set to MS:MS_COMMENTS 50 to 1500 m/z. Instrument parameters were as follows: gas, 200 °C at 4 L/min; MS:MS_COMMENTS nebulizer, 44 psi at 2000 V; sheath gas, 300 °C at 12 L/min, capillary, 3000 V; MS:MS_COMMENTS fragmentor, 100 V; skimmer, 65 V; and scan rate, 3 scans/second. The instrument MS:MS_COMMENTS was operated in negative ionization mode for all samples analyzed. Isotopologues MS:MS_COMMENTS and 13C enrichment in metabolites of interest were generated from analysis of MS:MS_COMMENTS the raw data #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Hepatocyte CM C13 Fructose 1 Hepatocyte CM C13 Fructose 2 Hepatocyte CM C13 Fructose 3 CaSki CM C13 Fructose 1 CaSki CM C13 Fructose 2 CaSki CM C13 Fructose 3 Factors Sample source:culture media | Conditioned media cell type:Hepatocytes Sample source:culture media | Conditioned media cell type:Hepatocytes Sample source:culture media | Conditioned media cell type:Hepatocytes Sample source:culture media | Conditioned media cell type:CaSki cells Sample source:culture media | Conditioned media cell type:CaSki cells Sample source:culture media | Conditioned media cell type:CaSki cells Fructose 15509061 14057536 14173209 13139699 11743921 11312989 Sorbitol 244091 243313 219755 242023 266051 238526 Glucose 2462097 2915844 2827902 0 0 0 Pyruvate 6893063 7779000 7778540 16450546 21161747 20984056 Lactic Acid 131836628 142800246 144650726 167806324 182577678 216287507 Aspartic Acid 374249 349232 287444 147025 199941 252491 Alanine 93816 96994 96517 1550742 521626 621445 cis-Aconitate 31424 42850 34599 60483 72061 77209 Malic acid 3065301 3276905 3600793 3605861 4221021 5145173 Glutamic Acid 2797055 3104159 1587614 3004677 3309227 4115213 Serine 74186 70015 65568 219277 239625 283614 Alpha-Ketoglutaric Acid 9695092 11054248 11058145 17495973 19086692 22497667 GABA 160742 182988 120544 189621 209198 257238 Succinic acid 103065 114408 111571 281085 336120 430826 Fumaric Acid 229582 245876 269597 271467 332310 430319 Uracil 128525 138755 125994 940351 1125575 1392506 Arginine 39767 41992 50264 34342 43612 46405 Citric acid 3335284 3014980 2841190 2802709 2920624 2601144 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG identifiers Fructose C00095 Sorbitol C00794 Glucose C00031 Pyruvate C00022 Lactic Acid C00186 Aspartic Acid C00049 Alanine C00041 cis-Aconitate C00417 Malic acid C00149 Glutamic Acid C00025 Serine C00065 Alpha-Ketoglutaric Acid C00026 GABA C00334 Succinic acid C00042 Fumaric Acid C00122 Uracil C00106 Arginine C00062 Citric acid C00158 METABOLITES_END #END