#METABOLOMICS WORKBENCH rjfowle_20241110_143449 DATATRACK_ID:5362 STUDY_ID:ST003571 ANALYSIS_ID:AN005866 PROJECT_ID:PR002203 VERSION 1 CREATED_ON November 12, 2024, 6:28 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 lipids that can metabolized by cancer cells ST:STUDY_SUMMARY Fructose is known to be metabolized by the liver to produce lipids that can ST:STUDY_SUMMARY circulate in the systemic circulation. Metabolism of fructose by CaSki cancer ST:STUDY_SUMMARY cells, on the other hand, is minimal. Therefore, we wished to understand if ST:STUDY_SUMMARY fructose metabolism in hepatocytes resulted in the release of lipids that CaSki ST:STUDY_SUMMARY cells could then uptake and metabolize to support their growth. Therefore, we ST:STUDY_SUMMARY formed fresh media which contained fructose as the only sugar source in the ST:STUDY_SUMMARY media. We cultured primary hepatocytes from C57BL/6 mice in this fresh media to ST:STUDY_SUMMARY form hepatocyte-conditioned media. We then transferred this ST:STUDY_SUMMARY hepatocyte-conditioned media to CaSki cells to form CaSki-conditioned media. ST:STUDY_SUMMARY LC-MS analysis on reverse-phase chromatography of fresh media, ST:STUDY_SUMMARY hepatocyte-conditioned media, and CaSki-conditioned media revealed that several ST:STUDY_SUMMARY lysophosphatidylcholine species are released from hepatocytes and up taken by ST:STUDY_SUMMARY 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 - Fresh media 1 Sample source:culture media | Conditioned media cell type:no cells RAW_FILE_NAME(Raw file name)=Fresh media 1.mzXML SUBJECT_SAMPLE_FACTORS - Fresh media 2 Sample source:culture media | Conditioned media cell type:no cells RAW_FILE_NAME(Raw file name)=Fresh media 2.mzXML SUBJECT_SAMPLE_FACTORS - Fresh media 3 Sample source:culture media | Conditioned media cell type:no cells RAW_FILE_NAME(Raw file name)=Fresh media 3.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 1 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 1.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 2 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 2.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 3 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 3.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 4 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 4.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 5 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 5.mzXML SUBJECT_SAMPLE_FACTORS - hepatocyte CM 6 Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes RAW_FILE_NAME(Raw file name)=hepatocyte CM 6.mzXML SUBJECT_SAMPLE_FACTORS - Caski CM 1 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=Caski CM 1.mzXML SUBJECT_SAMPLE_FACTORS - Caski CM 2 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=Caski CM 2.mzXML SUBJECT_SAMPLE_FACTORS - Caski CM 3 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=Caski CM 3.mzXML SUBJECT_SAMPLE_FACTORS - Caski CM 4 Sample source:culture media | Conditioned media cell type:CaSki cells RAW_FILE_NAME(Raw file name)=Caski CM 4.mzXML #COLLECTION CO:COLLECTION_SUMMARY 10 mM fructose was dissolved in glucose-free DMEM and 10% dialyzed FBS with 1% CO:COLLECTION_SUMMARY penicillin/streptomycin. This media with no cells composed the fresh media. 50 CO:COLLECTION_SUMMARY uL of this media was then collected and later extracted for LC-MS analysis, CO:COLLECTION_SUMMARY composing the fresh media condition. The media was then used to culture CO:COLLECTION_SUMMARY hepatocytes for 24 hours, yielding hepatocyte conditioned media (CM). 50 uL of CO:COLLECTION_SUMMARY this media was collected and later extracted for LC-MS analysis, composing the CO:COLLECTION_SUMMARY hepatocyte conditioned media collection. This hepatocyte conditioned media was CO:COLLECTION_SUMMARY then transferred to CaSki cells for 24 hours, yielding Caski conditioned media CO:COLLECTION_SUMMARY (CM). 50 uL of this media was collected and later extracted for LC-MS analysis, CO:COLLECTION_SUMMARY composing the CaSki conditioned media. CO:SAMPLE_TYPE Culture Media #TREATMENT TR:TREATMENT_SUMMARY 10 mM fructose was dissolved in glucose-free DMEM and 10% dialyzed FBS with 1% TR:TREATMENT_SUMMARY penicillin/streptomycin. This media with no cells composed the fresh media. The TR:TREATMENT_SUMMARY media was then used to culture hepatocytes for 24 hours, yielding hepatocyte TR:TREATMENT_SUMMARY conditioned media (CM). This hepatocyte conditioned media was then transferred TR:TREATMENT_SUMMARY to CaSki cells for 24 hours, yielding Caski conditioned media (CM). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For extractions of cell-culture media, 50 µL of serum was added to a Captiva SP:SAMPLEPREP_SUMMARY EMR 96 well plate (Agilent, Santa Clara, CA). Acetonitrile:methanol (1:1, 200 SP:SAMPLEPREP_SUMMARY µL) with labeled internal standards was added to the plate and incubated for 1 SP:SAMPLEPREP_SUMMARY minute on a plate shaker and at 4 °C for 10 minutes. Methaol:acetonitrile:water SP:SAMPLEPREP_SUMMARY (2:2:1, 150 µL) was added to the plate and eluted by using a positive pressure SP:SAMPLEPREP_SUMMARY manifold into a collection plate. The Captiva EMR 96-well plate was washed one SP:SAMPLEPREP_SUMMARY additional time with methanol:acetonitrile:water (2:2:1) and eluted for polar SP:SAMPLEPREP_SUMMARY metabolites. For nonpolar metabolites, a new collection plate was used, and SP:SAMPLEPREP_SUMMARY eluted with 1:1 methanol:methyl tert-butyl ether (MTBE). Eluted nonpolar SP:SAMPLEPREP_SUMMARY metabolites were dried under a stream of N2 by a Biotage N2-dryer. Nonpolar SP:SAMPLEPREP_SUMMARY metabolites were reconstituted in 1:1 isopropanol:methanol. A pooled-reference SP:SAMPLEPREP_SUMMARY sample was prepared by mixing aliquots of all samples prior to extraction. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed by using a HSS T3 column (Acquity; 150 × 2.1 mm, 1.8 μm) CH:CHROMATOGRAPHY_SUMMARY interfaced with an Agilent 6545 QTOF. The LC system used was an Agilent 1290 CH:CHROMATOGRAPHY_SUMMARY Infinity II. Mobile-phase solvents had the following composition: A = 60% CH:CHROMATOGRAPHY_SUMMARY acetonitrile, 40% water, 0.1% formic acid, 10 mM ammonium formate 2.5 μM CH:CHROMATOGRAPHY_SUMMARY medronic acid and B = 90% 2-propanol, 10% acetonitrile, 0.1% formic acid, 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium formate. The following linear gradient was used: 0-2 min, 30% B; 17 CH:CHROMATOGRAPHY_SUMMARY min, 75% B; 20 min, 85% B; 23-26 min, 100% B; 26 min, 30% B. Injection volumes CH:CHROMATOGRAPHY_SUMMARY were 4 μL. The column compartment was maintained at 60 °C CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate; 2.5 μM CH:SOLVENT_A medronic acid CH:SOLVENT_B 90% 2-propanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate CH:FLOW_GRADIENT 0-2 min, 30% B; 17 min, 75% B; 20 min, 85% B; 23-26 min, 100% B; 26 min, 30% B CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS LC/MS of nonpolar metabolites was performed by using a HSS T3 column (Acquity; MS:MS_COMMENTS 150 × 2.1 mm, 1.8 μm) interfaced with an Agilent 6545 Q-TOF. The mass range MS:MS_COMMENTS was 120-1200 m/z. Instrument parameters were as follows: gas, 250°C at 11 MS:MS_COMMENTS L/min; nebulizer pressure, 35 psi; sheath gas temperature, 300°C; sheath gas MS:MS_COMMENTS flow 12 L/min; VCap 3000 V; nozzle voltage 500 V; Fragmentor 160 V; Skimmer 65 MS:MS_COMMENTS V. The instrument was operated in positive ionization mode for all samples MS:MS_COMMENTS analyzed. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Fresh media 1 Fresh media 2 Fresh media 3 hepatocyte CM 1 hepatocyte CM 2 hepatocyte CM 3 hepatocyte CM 4 hepatocyte CM 5 hepatocyte CM 6 Caski CM 1 Caski CM 2 Caski CM 3 Caski CM 4 Factors Sample source:culture media | Conditioned media cell type:no cells Sample source:culture media | Conditioned media cell type:no cells Sample source:culture media | Conditioned media cell type:no cells Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes Sample source:culture media | Conditioned media cell type:mouse primary hepatocytes Sample source:culture media | Conditioned media cell type:mouse primary 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 Sample source:culture media | Conditioned media cell type:CaSki cells ACar 14:2 6646 6260 7268 4334 5203 4402 4017 5070 4558 5246 4230 4171 6007 LPC 0:0/18:0 471 823 909 1416 738 930 124 108 512 0 171 0 0 LPC 0:0/18:1 8757 9428 9280 7934 4742 5836 6227 5925 4814 445 836 359 697 LPC 0:0/18:2 5212 4224 5036 12371 9714 9044 11635 11276 9332 2623 3748 3538 3698 LPC 0:0/20:4 5061 3535 4648 11882 7323 7389 11058 11973 8565 0 0 0 0 LPC 0:0/22:6 871 658 400 6807 7508 6759 6772 7106 5189 0 0 0 0 LPC 14:0/0:0 0 317 259 0 0 0 0 0 0 0 0 0 0 LPC 18:1/0:0 8757 9286 9032 7741 4557 5424 6227 5925 4814 445 836 359 697 LPC 18:2/0:0 5212 4224 5036 12371 9164 8786 11635 11276 9271 2618 3748 3537 3706 LPC 20:4/0:0 5031 3535 4648 11743 8796 8620 11163 11864 8548 0 0 0 0 LPC 22:6/0:0 911 697 502 9855 7011 6582 8256 9229 6718 0 0 0 0 TG 16:0_16:0_18:1 1941 2376 1706 4323 3101 2931 3295 3465 3373 3162 4315 3667 3559 TG 16:0_16:1_18:1 5715 7659 4948 16280 11025 11517 11409 13833 11403 12448 15128 14463 12323 TG 16:0_18:0_18:1 1217 2081 1292 4860 3267 3133 3160 3912 3529 3420 5675 4763 3463 TG 16:0_18:1_22:0 827 1170 678 2962 2123 1699 1783 2352 2295 2167 3093 2737 2339 TG 18:0_18:1_18:1 799 1646 886 3540 2199 2451 2325 2698 2886 2799 5070 4177 2860 TG 18:1_18:2_22:0 0 0 0 2202 881 1079 954 1206 753 1038 1563 1635 1103 TG 50:3 465 685 268 5139 2890 3134 3293 4578 3253 3226 4054 3484 3611 TG 52:2 1550 2177 1079 5162 3381 3327 3102 3685 3936 3975 5404 4725 3867 TG 52:3 233 2260 497 6179 3788 3994 4106 3697 3449 4147 4268 4410 3896 TG 56:2 108 106 0 2775 1340 1580 1548 1862 1455 1596 1987 1974 1630 TG 56:8 0 0 0 4651 2410 2725 2329 2725 1971 2344 2774 2862 2464 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Exact mass ACar 14:2 367.2723 LPC 0:0/18:0 523.3638 LPC 0:0/18:1 521.3481 LPC 0:0/18:2 519.3325 LPC 0:0/20:4 543.3325 LPC 0:0/22:6 567.3325 LPC 14:0/0:0 467.3012 LPC 18:1/0:0 521.3481 LPC 18:2/0:0 519.3325 LPC 20:4/0:0 543.3325 LPC 22:6/0:0 567.3325 TG 16:0_16:0_18:1 832.752 TG 16:0_16:1_18:1 830.7363 TG 16:0_18:0_18:1 860.7833 TG 16:0_18:1_22:0 916.8459 TG 18:0_18:1_18:1 886.7989 TG 18:1_18:2_22:0 940.8459 TG 50:3 828.7207 TG 52:2 858.7676 TG 52:3 856.752 TG 56:2 914.8302 TG 56:8 902.7363 METABOLITES_END #END