#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