#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