#METABOLOMICS WORKBENCH juliehaines_20240501_131926 DATATRACK_ID:4808 STUDY_ID:ST003184 ANALYSIS_ID:AN005230 PROJECT_ID:PR001983 VERSION 1 CREATED_ON May 2, 2024, 6:17 pm #PROJECT PR:PROJECT_TITLE A multimodal drug-diet-immunotherapy combination restrains melanoma progression PR:PROJECT_TITLE and metastasis PR:PROJECT_SUMMARY The genetic landscape of cancer cells can lead to specific metabolic PR:PROJECT_SUMMARY dependencies for tumor growth. Dietary interventions represent an attractive PR:PROJECT_SUMMARY strategy to restrict the availability of key nutrients to tumors. In this study, PR:PROJECT_SUMMARY we identified that growth of a subset of melanoma was severely restricted by a PR:PROJECT_SUMMARY rationally designed combination therapy of a stearoyl-CoA desaturase (SCD) PR:PROJECT_SUMMARY inhibitor with an isocaloric low-oleic acid diet. Despite its importance in PR:PROJECT_SUMMARY oncogenesis, SCD underwent monoallelic co-deletion along with PTEN on chromosome PR:PROJECT_SUMMARY 10q in about 47.5% of melanoma, and the other SCD allele was methylated, PR:PROJECT_SUMMARY resulting in very low SCD expression. While this SCD deficient subset was PR:PROJECT_SUMMARY refractory to SCD inhibitors, the subset of PTEN wildtype melanoma that retained PR:PROJECT_SUMMARY SCD was sensitive. As dietary oleic acid could potentially blunt the effect of PR:PROJECT_SUMMARY SCD inhibitors, a low-oleic acid custom diet was combined with SCD inhibitor. PR:PROJECT_SUMMARY The combination reduced monounsaturated fatty acids and increased saturated PR:PROJECT_SUMMARY fatty acids, inducing robust apoptosis and growth suppression and inhibiting PR:PROJECT_SUMMARY lung metastasis with minimal toxicity in preclinical mouse models of PTEN PR:PROJECT_SUMMARY wildtype melanoma. When combined with anti-PD1 immunotherapy, the SCD inhibitor PR:PROJECT_SUMMARY improved T cell functionality and further constrained melanoma growth in mice. PR:PROJECT_SUMMARY Collectively, these results suggest that optimizing SCD inhibitors with diets PR:PROJECT_SUMMARY low in oleic acid may offer a viable and efficacious therapeutic approach for PR:PROJECT_SUMMARY improving melanoma treatment. PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Biplab Dasgupta PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE A multimodal drug-diet-immunotherapy combination restrains melanoma progression ST:STUDY_TITLE and metastasis - tumor lipidomics ST:STUDY_SUMMARY Lipidomics profiling of immunocompetent B16F10 model of melanoma to examine ST:STUDY_SUMMARY lipid levels in tumors following reduced oleic acid content in diet combined ST:STUDY_SUMMARY with the stearyl CoA desaturase inhibitor CAY10566. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:GENDER Not applicable #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 - DS2-039-001 factor:Vehicle+ low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-001 SUBJECT_SAMPLE_FACTORS - DS2-039-002 factor:Vehicle+ low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-002 SUBJECT_SAMPLE_FACTORS - DS2-039-003 factor:Vehicle+ low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-003 SUBJECT_SAMPLE_FACTORS - DS2-039-004 factor:Vehicle+ low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-004 SUBJECT_SAMPLE_FACTORS - DS2-039-005 factor:Vehicle+ low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-005 SUBJECT_SAMPLE_FACTORS - DS2-039-006 factor:SCD inhibitor + low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-006 SUBJECT_SAMPLE_FACTORS - DS2-039-007 factor:SCD inhibitor + low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-007 SUBJECT_SAMPLE_FACTORS - DS2-039-008 factor:SCD inhibitor + low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-008 SUBJECT_SAMPLE_FACTORS - DS2-039-009 factor:SCD inhibitor + low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-009 SUBJECT_SAMPLE_FACTORS - DS2-039-010 factor:SCD inhibitor + low oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-010 SUBJECT_SAMPLE_FACTORS - DS2-039-011 factor:Vehicle + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-011 SUBJECT_SAMPLE_FACTORS - DS2-039-012 factor:Vehicle + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-012 SUBJECT_SAMPLE_FACTORS - DS2-039-013 factor:Vehicle + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-013 SUBJECT_SAMPLE_FACTORS - DS2-039-014 factor:Vehicle + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-014 SUBJECT_SAMPLE_FACTORS - DS2-039-015 factor:Vehicle + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-015 SUBJECT_SAMPLE_FACTORS - DS2-039-016 factor:SCD inhibitor + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-016 SUBJECT_SAMPLE_FACTORS - DS2-039-017 factor:SCD inhibitor + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-017 SUBJECT_SAMPLE_FACTORS - DS2-039-018 factor:SCD inhibitor + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-018 SUBJECT_SAMPLE_FACTORS - DS2-039-019 factor:SCD inhibitor + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-019 SUBJECT_SAMPLE_FACTORS - DS2-039-020 factor:SCD inhibitor + high oleic acid diet | Sample source:tumor RAW_FILE_NAME(raw file name)=DS2-039-020 #COLLECTION CO:COLLECTION_SUMMARY For immune checkpoint inhibitor (ICI) studies, 1x10^6 B16F10 cells were mixed CO:COLLECTION_SUMMARY with matrigel 1:1 and implanted subcutaneously in each flank into C57BL/6 mice. CO:COLLECTION_SUMMARY Mice were randomized into groups with combinations of sunflower or coconut oil CO:COLLECTION_SUMMARY diet, vehicle or CAY10566, and IgG or anti-PD1 treatments. CAY10566 was prepared CO:COLLECTION_SUMMARY and administered as previously described. IgG and anti-PD1 were diluted in PBS CO:COLLECTION_SUMMARY to a concentration of 1ug/uL. 100uL of IgG or anti-PD1 were administered CO:COLLECTION_SUMMARY intraperitoneally twice weekly, every 3-4 days. Mice were euthanized and tumors CO:COLLECTION_SUMMARY were harvested 17 days after tumor implantation/14 days after the start of CO:COLLECTION_SUMMARY treatment. Tumor tissues were processed for flow cytometry analysis. For lung CO:COLLECTION_SUMMARY metastasis studies, ZsGreen expressing B16F10 cells were established by CO:COLLECTION_SUMMARY transducing them with concentrated ZsGreen virus produced by the Viral Vector CO:COLLECTION_SUMMARY Core at CCHMC. Cells were then FACS sorted to obtain a pure population of CO:COLLECTION_SUMMARY B16F10-ZsGreen cells prior to injection. 2x10^5 B16F10-ZsGreen cells were CO:COLLECTION_SUMMARY injected via tail vein into C57BL/6 mice by trained veterinary staff of CCHMC. CO:COLLECTION_SUMMARY Mice were randomized into groups with combinations of sunflower or coconut oil CO:COLLECTION_SUMMARY diet, and vehicle or CAY10566. CAY10566 was prepared and administered as CO:COLLECTION_SUMMARY previously described. Mice were euthanized and lungs were harvested 17 days CO:COLLECTION_SUMMARY after tumor cell injection/14 days after the start of treatment. B16F10-ZsGreen CO:COLLECTION_SUMMARY metastatic tumor nodes were detected by fluorescence microscopy, and CO:COLLECTION_SUMMARY quantification was performed using Image J. CO:SAMPLE_TYPE Tumor cells #TREATMENT TR:TREATMENT_SUMMARY All animal procedures were carried out in accordance with the IACUC-approved TR:TREATMENT_SUMMARY protocol of Cincinnati Children’s Hospital Medical Center. Animals were TR:TREATMENT_SUMMARY monitored daily by Veterinary Services. For subcutaneous tumor implantation, TR:TREATMENT_SUMMARY 1x10^6 B16F10 cells were mixed with matrigel 1:1 and implanted subcutaneously TR:TREATMENT_SUMMARY into NOD-SCID IL2Rgnull or C57BL/6 mice, respectively. CAY10566 was suspended in TR:TREATMENT_SUMMARY 0.5% Methyl Cellulose in water with 0.2% Tween 80 at 9 parts per 1 part DMSO TR:TREATMENT_SUMMARY stock. CAY10566 was administered at 30mg/kg via oral gavage, twice daily, with a TR:TREATMENT_SUMMARY drug holiday during the weekends. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For tumor analysis, tissue was weighed and placed into 1.5mL Eppendorf Safe-Lock SP:SAMPLEPREP_SUMMARY tubes that were pre-filled with 1.0mm glass grinder beads (Biospec Products SP:SAMPLEPREP_SUMMARY Inc). Cold methanol was then added to each tube so that all final tissue SP:SAMPLEPREP_SUMMARY concentrations were 20 mg/mL. Samples were then homogenized via a “Next SP:SAMPLEPREP_SUMMARY Advance” Bullet Blender for 5 minutes and subsequently vortexed for 30 SP:SAMPLEPREP_SUMMARY minutes. Following vortex, insoluble material was pelleted by centrifugation SP:SAMPLEPREP_SUMMARY (18000g, 10 min) and supernatants were isolated for analysis by UHPLC-MS. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative C18 fatty acids CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 75% water/25% acetonitrile; 5 mM ammonium acetate CH:SOLVENT_B 50% isopropanol/45% acetonitrile; 5 mM ammonium acetate CH:FLOW_GRADIENT 0-1 min 25% B and 0.3 ml/min; 1-2 min 25-50% B and 0.3 mL/min; 2–8 min CH:FLOW_GRADIENT 50–90% B and 0.3 mL/min; 8–10 min 90–99% B and 0.3 mL/min; 10–14 min CH:FLOW_GRADIENT hold at 99% B and 0.3 mL/min; 14–14.1 min 99–25% B and 0.3 mL/min; CH:FLOW_GRADIENT 14.1–16.9 min hold at 25% B and 0.4 mL/min; 16.9–17 min hold at 25% B and CH:FLOW_GRADIENT resume flow of 0.3 mL/min CH:FLOW_RATE 0.3-0.4 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Q Exactive was run independently in positive and negative ion mode, scanning MS:MS_COMMENTS using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS:MS_COMMENTS MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb MS:MS_COMMENTS sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was MS:MS_COMMENTS performed prior to the run using the PierceTM Positive and Negative Ion MS:MS_COMMENTS Calibration Solutions (Thermo Fisher Scientific). Run order of samples was MS:MS_COMMENTS randomized and technical replicates were injected after every 4 samples to MS:MS_COMMENTS assess quality control. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples DS2-039-001 DS2-039-002 DS2-039-003 DS2-039-004 DS2-039-005 DS2-039-006 DS2-039-007 DS2-039-008 DS2-039-009 DS2-039-010 DS2-039-011 DS2-039-012 DS2-039-013 DS2-039-014 DS2-039-015 DS2-039-016 DS2-039-017 DS2-039-018 DS2-039-019 DS2-039-020 Factors factor:Vehicle+ low oleic acid diet | Sample source:tumor factor:Vehicle+ low oleic acid diet | Sample source:tumor factor:Vehicle+ low oleic acid diet | Sample source:tumor factor:Vehicle+ low oleic acid diet | Sample source:tumor factor:Vehicle+ low oleic acid diet | Sample source:tumor factor:SCD inhibitor + low oleic acid diet | Sample source:tumor factor:SCD inhibitor + low oleic acid diet | Sample source:tumor factor:SCD inhibitor + low oleic acid diet | Sample source:tumor factor:SCD inhibitor + low oleic acid diet | Sample source:tumor factor:SCD inhibitor + low oleic acid diet | Sample source:tumor factor:Vehicle + high oleic acid diet | Sample source:tumor factor:Vehicle + high oleic acid diet | Sample source:tumor factor:Vehicle + high oleic acid diet | Sample source:tumor factor:Vehicle + high oleic acid diet | Sample source:tumor factor:Vehicle + high oleic acid diet | Sample source:tumor factor:SCD inhibitor + high oleic acid diet | Sample source:tumor factor:SCD inhibitor + high oleic acid diet | Sample source:tumor factor:SCD inhibitor + high oleic acid diet | Sample source:tumor factor:SCD inhibitor + high oleic acid diet | Sample source:tumor factor:SCD inhibitor + high oleic acid diet | Sample source:tumor C16:0 FA 3946499328 4244123392 6434538496 3878599168 4095853056 4197381888 4411759104 4451825664 8484619264 3807301632 3393046528 11778848768 6657183744 4172298752 4352745984 6062859264 5495313920 3348405504 3971208192 3416923392 C18:0 FA 2492192512 2929144320 5485698560 2636876800 2763056896 3707086080 3597035776 4381108736 6443791872 3611325184 2329693184 10773140480 5570484736 2607196672 3134503680 4660334592 4831761920 3081509888 3631853312 3032824576 C18:1 FA 5929195008 6062060032 9972813824 5035459584 4479164416 1346573312 1236686976 1451869824 3417352704 1023947520 4027694080 19271225344 9621735424 4629622784 5875468288 3457213440 3184717056 1545557376 1857017088 1703752832 C18:2 FA 2150335744 1398792960 2825823232 1286434816 1807587712 1766256384 1735987584 2253646848 5705310720 1373085952 1051265216 7569892864 4040301824 1266965888 1985753088 3171220736 2362421760 1281456768 1946000640 1623146624 C16:3 FA 3627552 1090362 3829161 1750069 3152372 1723729 1319629 1770602 6880750 2794126 2134414 5657748 3277953 2182908 3303442 3877603 1701427 2464689 2263920 2648793 α-C18:3 FA 46593428 14453279 33306526 11985863 20566742 14794645 14484087 23540880 48358380 16172380 18455276 80849792 38711284 17662208 28913574 26531176 34107000 10367993 16715937 15755033 γ-C18:3 FA 53523712 19394638 39635076 26629374 23247422 46731764 56669328 67862872 322636128 66890176 24283024 52184236 37656300 25288030 41105688 152896032 86943528 43889648 40442084 34315820 C16:1 FA 1046261632 776501568 1405359232 806883136 727403840 216385936 214149024 233196912 862752128 232321408 607769664 1954457600 898908544 696762240 759337792 508595488 320681792 204765408 255146016 201985872 C16:2 FA 19918312 10313996 22368968 16186549 17655246 5192404 3709662 7356835 24180790 6206628 12168036 40836332 17683136 14550474 19032312 15416025 7790646 5447898 10591360 8396791 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Lipid Class m/z rt C16:0 FA FA 255.232742 8.839 C18:0 FA FA 283.264374 10.127 C18:1 FA FA 281.248566 9.094 C18:2 FA FA 279.232941 8.101 C16:3 FA FA 249.186172 5.964 α-C18:3 FA FA 277.217346 7.188 γ-C18:3 FA FA 277.217438 7.318 C16:1 FA FA 253.217117 7.727 C16:2 FA FA 251.201523 6.741 METABOLITES_END #END