#METABOLOMICS WORKBENCH Izar_Lab_20230831_233029 DATATRACK_ID:4278 STUDY_ID:ST002851 ANALYSIS_ID:AN004671 PROJECT_ID:PR001785
VERSION             	1
CREATED_ON             	September 6, 2023, 11:19 pm
#PROJECT
PR:PROJECT_TITLE                 	A genetic-metabolic axis confers liver-metastatic organotropism
PR:PROJECT_SUMMARY               	Transcriptomic and metabolomic analyses in animals revealed distinct metabolic
PR:PROJECT_SUMMARY               	adaptations, particularly related to the TCA cycle and OxPhos, specific to liver
PR:PROJECT_SUMMARY               	metastases compared to concurrent lung metastases. This finding was
PR:PROJECT_SUMMARY               	substantiated by analyzing RNA-seq data from a considerable number of patient
PR:PROJECT_SUMMARY               	metastases across various cancer types. Further analysis of exome/RNA-seq data
PR:PROJECT_SUMMARY               	from melanoma patients indicated more frequent PIK3CA mutations, lower
PR:PROJECT_SUMMARY               	transcript levels of PIP4K2C, and enrichment of TCA cycle and OxPhos pathways in
PR:PROJECT_SUMMARY               	liver metastases.
PR:INSTITUTE                     	Columbia University - Medical Center
PR:DEPARTMENT                    	Department of Medicine
PR:LABORATORY                    	Division of Hematology/Oncology
PR:LAST_NAME                     	Izar
PR:FIRST_NAME                    	Benjamin
PR:ADDRESS                       	Columbia University Irving Medical Center, New York, NY, USA
PR:EMAIL                         	bi2175@cumc.columbia.edu
PR:PHONE                         	0123456789
#STUDY
ST:STUDY_TITLE                   	Metabolic caracterization of liver metastasis organotropism
ST:STUDY_SUMMARY                 	Transcriptomic and metabolomic analyses in animals revealed distinct metabolic
ST:STUDY_SUMMARY                 	adaptations, particularly related to the TCA cycle and OxPhos, specific to liver
ST:STUDY_SUMMARY                 	metastases compared to concurrent lung metastases. This finding was
ST:STUDY_SUMMARY                 	substantiated by analyzing RNA-seq data from a considerable number of patient
ST:STUDY_SUMMARY                 	metastases across various cancer types. Further analysis of exome/RNA-seq data
ST:STUDY_SUMMARY                 	from melanoma patients indicated more frequent PIK3CA mutations, lower
ST:STUDY_SUMMARY                 	transcript levels of PIP4K2C, and enrichment of TCA cycle and OxPhos pathways in
ST:STUDY_SUMMARY                 	liver metastases.
ST:INSTITUTE                     	Columbia University
ST:DEPARTMENT                    	Department of Medicine
ST:LABORATORY                    	Division of Hematology/Oncology
ST:LAST_NAME                     	Izar
ST:FIRST_NAME                    	Benjamin
ST:ADDRESS                       	Columbia University Irving Medical Center, New York, NY, USA
ST:EMAIL                         	bi2175@cumc.columbia.edu
ST:PHONE                         	0123456789
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#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           	-	WT_Lung_MR01	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Lung_MR01
SUBJECT_SAMPLE_FACTORS           	-	WT_Lung_MR02	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Lung_MR02
SUBJECT_SAMPLE_FACTORS           	-	WT_Lung_MR03	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Lung_MR03
SUBJECT_SAMPLE_FACTORS           	-	WT_Lung_MR04	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Lung_MR04
SUBJECT_SAMPLE_FACTORS           	-	WT_Lung_MR05	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Lung_MR05
SUBJECT_SAMPLE_FACTORS           	-	WT_Liver_MR06	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Liver_MR06
SUBJECT_SAMPLE_FACTORS           	-	WT_Liver_MR07	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Liver_MR07
SUBJECT_SAMPLE_FACTORS           	-	WT_Liver_MR08	Organism:Mus musculus | Organism part:lung | Variant:WT NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=WT_Liver_MR08
SUBJECT_SAMPLE_FACTORS           	-	KO_Lung_MR09	Organism:Mus musculus | Organism part:lung | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Lung_MR09
SUBJECT_SAMPLE_FACTORS           	-	KO_Lung_MR10	Organism:Mus musculus | Organism part:lung | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Lung_MR10
SUBJECT_SAMPLE_FACTORS           	-	KO_Lung_MR11	Organism:Mus musculus | Organism part:lung | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Lung_MR11
SUBJECT_SAMPLE_FACTORS           	-	KO_Lung_MR12	Organism:Mus musculus | Organism part:lung | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Lung_MR12
SUBJECT_SAMPLE_FACTORS           	-	KO_Lung_MR13	Organism:Mus musculus | Organism part:lung | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Lung_MR13
SUBJECT_SAMPLE_FACTORS           	-	KO_Liver_MR14	Organism:Mus musculus | Organism part:liver | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Liver_MR14
SUBJECT_SAMPLE_FACTORS           	-	KO_Liver_MR15	Organism:Mus musculus | Organism part:liver | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Liver_MR15
SUBJECT_SAMPLE_FACTORS           	-	KO_Liver_MR16	Organism:Mus musculus | Organism part:liver | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Liver_MR16
SUBJECT_SAMPLE_FACTORS           	-	KO_Liver_MR17	Organism:Mus musculus | Organism part:liver | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Liver_MR17
SUBJECT_SAMPLE_FACTORS           	-	KO_Liver_MR18	Organism:Mus musculus | Organism part:liver | Variant:KN NOD/SCID/IL2gR	Sample type=experimental sample; RAW_FILE_NAME=KO_Liver_MR18
SUBJECT_SAMPLE_FACTORS           	-	QC_Mix_001	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Mix_001
SUBJECT_SAMPLE_FACTORS           	-	QC_Mix_002	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Mix_002
SUBJECT_SAMPLE_FACTORS           	-	QC_Mix_003	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Mix_003
SUBJECT_SAMPLE_FACTORS           	-	QC_Lung_001	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Lung_001
SUBJECT_SAMPLE_FACTORS           	-	QC_Lung_002	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Lung_002
SUBJECT_SAMPLE_FACTORS           	-	QC_Lung_003	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Lung_003
SUBJECT_SAMPLE_FACTORS           	-	QC_Liver_001	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Liver_001
SUBJECT_SAMPLE_FACTORS           	-	QC_Liver_002	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Liver_002
SUBJECT_SAMPLE_FACTORS           	-	QC_Liver_003	Organism:Mus musculus | Organism part:control | Variant:-	Sample type=pooled quality control sample; RAW_FILE_NAME=QC_Liver_003
SUBJECT_SAMPLE_FACTORS           	-	ExtractionBlank_001	Organism:blank | Organism part:blank | Variant:-	Sample type=experimental blank; RAW_FILE_NAME=ExtractionBlank_001
SUBJECT_SAMPLE_FACTORS           	-	ExtractionBlank_002	Organism:blank | Organism part:blank | Variant:-	Sample type=experimental blank; RAW_FILE_NAME=ExtractionBlank_002
SUBJECT_SAMPLE_FACTORS           	-	ExtractionBlank_003	Organism:blank | Organism part:blank | Variant:-	Sample type=experimental blank; RAW_FILE_NAME=ExtractionBlank_003
#COLLECTION
CO:COLLECTION_SUMMARY            	Briefly, 6-8-week-old female NOD/SCID/IL2gR mice (005557 Jackson Laboratory)
CO:COLLECTION_SUMMARY            	were injected via tail vein with 1 x 10^5 A375 human melanoma cell line of
CO:COLLECTION_SUMMARY            	indicated genotypes, n=5/group. Mice were monitored over 3-4 weeks. When mice
CO:COLLECTION_SUMMARY            	showed signs of sickness, 20% of weight loss, impaired activity, hunching and
CO:COLLECTION_SUMMARY            	limb paralysis, animals were euthanized, and organs were harvested for
CO:COLLECTION_SUMMARY            	evaluation on metastasis presence, liver and lung metastasis were snap frozen
CO:COLLECTION_SUMMARY            	and subjected to downstream analysis.
CO:SAMPLE_TYPE                   	Biopsy
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Briefly, 6-8-week-old female NOD/SCID/IL2gR mice (005557 Jackson Laboratory)
TR:TREATMENT_SUMMARY             	were injected via tail vein with 1 x 10^5 A375 human melanoma cell line of
TR:TREATMENT_SUMMARY             	indicated genotypes, n=5/group.
TR:ANIMAL_ENDP_EUTHANASIA        	When mice showed signs of sickness, 20% of weight loss, impaired activity,
TR:ANIMAL_ENDP_EUTHANASIA        	hunching and limb paralysis.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites were extracted by a two-step liquid extraction adapted from Sellik
SP:SAMPLEPREP_SUMMARY            	et al.[1], 50 mg of frozen tissue was used and 500 µL of prechilled methanol
SP:SAMPLEPREP_SUMMARY            	(-80 °C) was added. For quantification and data normalization, 25 µL of
SP:SAMPLEPREP_SUMMARY            	internal standard solution containing 13C6-L-Arginine, 13C5 L Valine,
SP:SAMPLEPREP_SUMMARY            	13C2-Citric acid, 2H4-Succinic acid and 13C6-Fructose-6-phosphate at a
SP:SAMPLEPREP_SUMMARY            	concentration of 100 µM was added. This results in a concentration of 10 µM in
SP:SAMPLEPREP_SUMMARY            	the final extract. The sample was homogenized by a tissue slicer and the extract
SP:SAMPLEPREP_SUMMARY            	was vortexed for 2 min until no larger cell clusters were visible. Afterward,
SP:SAMPLEPREP_SUMMARY            	the sample was sonicated for 2 min in a chilled (0 °C) ultrasonic bath. The
SP:SAMPLEPREP_SUMMARY            	cells were kept at -80 °C for 5 min and subsequently thawed. The thawed cells
SP:SAMPLEPREP_SUMMARY            	were vortexed and sonicated for 2 min each and centrifuged at 3000 x g for 5
SP:SAMPLEPREP_SUMMARY            	min. The supernatant was collected and 250 µL of water acidified with 0.1 %
SP:SAMPLEPREP_SUMMARY            	acetic acid (LC-MS grade) was added. The sample was vortexed and sonicated for 2
SP:SAMPLEPREP_SUMMARY            	min and subject to the same freezing-thawing cycle described before. The samples
SP:SAMPLEPREP_SUMMARY            	were centrifuged at 3000 x g for 5 min and the supernatant was collected. The
SP:SAMPLEPREP_SUMMARY            	combined supernatant was dried in a vacuum-centrifuge for 45 min. The residue
SP:SAMPLEPREP_SUMMARY            	was resuspended in 250 µL acetonitrile/water (50/50; v/v) by sonicating and
SP:SAMPLEPREP_SUMMARY            	vortexing for 2 min each. To remove remaining proteins or other cell debris the
SP:SAMPLEPREP_SUMMARY            	extracts have been centrifuged at 12,000 rpm for 2 min and filtered using
SP:SAMPLEPREP_SUMMARY            	Nanosep 3KDa Omega centrifugal filters (Pall, Port Washington, USA). After the
SP:SAMPLEPREP_SUMMARY            	extraction pooled QC samples were generated for the whole sample set as well as
SP:SAMPLEPREP_SUMMARY            	for each investigated sample group and analyzed along the study samples to
SP:SAMPLEPREP_SUMMARY            	monitor the analytical quality. Ref: [1] (Sellick CA, Hansen R, Stephens GM,
SP:SAMPLEPREP_SUMMARY            	Goodacre R, Dickson AJ. Metabolite extraction from suspension-cultured mammalian
SP:SAMPLEPREP_SUMMARY            	cells for global metabolite profiling. Nat Protoc. 2011 28;6(8):1241-9. doi:
SP:SAMPLEPREP_SUMMARY            	10.1038/nprot.2011.366)
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	For liquid chromatography, an Agilent 1290 Infinity II Bio LC system (Agilent
CH:CHROMATOGRAPHY_SUMMARY        	Technologies Inc., Waldbronn, Germany) consisting of a 1290 Bio High-Speed Pump,
CH:CHROMATOGRAPHY_SUMMARY        	a 1290 Bio Multisampler and a 1290 MCT with an AdvanceBio MS Spent Media (150 x
CH:CHROMATOGRAPHY_SUMMARY        	2.1 mm, 2.7 µm; Agilent Technologies Inc., Waldbronn, Germany) were used. The
CH:CHROMATOGRAPHY_SUMMARY        	elution was carried out by utilizing a gradient at a flow rate of 250 μL/min
CH:CHROMATOGRAPHY_SUMMARY        	with water (10 mM ammonium acetate pH 9) as solvent A and acetonitrile/water
CH:CHROMATOGRAPHY_SUMMARY        	(90/10; v/v; 10 mM ammonium acetate pH 9) as solvent B. The linear gradient was:
CH:CHROMATOGRAPHY_SUMMARY        	0 min, 90% B; 2 min, 90% B; 12 min, 40% B; 13 min, 20% B; 16 min, 20% B;
CH:CHROMATOGRAPHY_SUMMARY        	followed by 9 min at initial condition for re-equilibration. Column temperature
CH:CHROMATOGRAPHY_SUMMARY        	was 35 °C, injection volume 5 µL.
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II Bio LC system
CH:COLUMN_NAME                   	AdvanceBio MS Spent Media (150 x 2.1 mm, 2.7 µm)
CH:SOLVENT_A                     	water (10 mM ammonium acetate pH 9)
CH:SOLVENT_B                     	acetonitrile/water (90/10; v/v; 10 mM ammonium acetate pH 9)
CH:FLOW_GRADIENT                 	none
CH:FLOW_RATE                     	250 μL/min
CH:COLUMN_TEMPERATURE            	35 °C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Data were analyszed in both positive and negative ion mode separately. MS-Dial
MS:MS_COMMENTS                   	version 4.9 was used for feature analysis.
MS:MS_RESULTS_FILE               	ST002851_AN004671_Results.txt	UNITS:Peak Area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END