#METABOLOMICS WORKBENCH juliehaines_20240502_073341 DATATRACK_ID:4811 STUDY_ID:ST003185 ANALYSIS_ID:AN005233 PROJECT_ID:PR001983 VERSION 1 CREATED_ON May 3, 2024, 10:13 am #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 - plasma lipidomics ST:STUDY_SUMMARY Lipidomics profiling of immunocompetent B16F10 model of melanoma to examine ST:STUDY_SUMMARY lipid levels in mouse plasma following reduced oleic acid content in diet ST:STUDY_SUMMARY combined 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 - DS2-039-021 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-021 SUBJECT_SAMPLE_FACTORS - DS2-039-022 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-022 SUBJECT_SAMPLE_FACTORS - DS2-039-023 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-023 SUBJECT_SAMPLE_FACTORS - DS2-039-024 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-024 SUBJECT_SAMPLE_FACTORS - DS2-039-025 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-025 SUBJECT_SAMPLE_FACTORS - DS2-039-026 factor:vehicle + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-026 SUBJECT_SAMPLE_FACTORS - DS2-039-027 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-027 SUBJECT_SAMPLE_FACTORS - DS2-039-028 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-028 SUBJECT_SAMPLE_FACTORS - DS2-039-029 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-029 SUBJECT_SAMPLE_FACTORS - DS2-039-030 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-030 SUBJECT_SAMPLE_FACTORS - DS2-039-031 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-031 SUBJECT_SAMPLE_FACTORS - DS2-039-032 factor:SCD inhibitor + low oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-032 SUBJECT_SAMPLE_FACTORS - DS2-039-033 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-033 SUBJECT_SAMPLE_FACTORS - DS2-039-034 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-034 SUBJECT_SAMPLE_FACTORS - DS2-039-035 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-035 SUBJECT_SAMPLE_FACTORS - DS2-039-036 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-036 SUBJECT_SAMPLE_FACTORS - DS2-039-037 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-037 SUBJECT_SAMPLE_FACTORS - DS2-039-038 factor:Vehicle + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-038 SUBJECT_SAMPLE_FACTORS - DS2-039-039 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-039 SUBJECT_SAMPLE_FACTORS - DS2-039-040 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-040 SUBJECT_SAMPLE_FACTORS - DS2-039-041 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-041 SUBJECT_SAMPLE_FACTORS - DS2-039-042 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-042 SUBJECT_SAMPLE_FACTORS - DS2-039-043 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-043 SUBJECT_SAMPLE_FACTORS - DS2-039-044 factor:SCD inhibitor + high oleic acid diet | Sample source:plasma RAW_FILE_NAME(raw file name)=DS2-039-044 #COLLECTION CO:COLLECTION_SUMMARY After two weeks of treatment, retroorbital whole blood samples were collected CO:COLLECTION_SUMMARY following Isoflurane sedation. The samples were obtained using heparinized CO:COLLECTION_SUMMARY capillary tubes (Catalog Number: 22-362566). Subsequently, these samples were CO:COLLECTION_SUMMARY placed into tubes containing K2E (Catalog Number: 365974), with a bulb used to CO:COLLECTION_SUMMARY drain the capillary tube (Catalog Number: 22-362566). The tubes were centrifuged CO:COLLECTION_SUMMARY for 15 minutes at 2,000 x g (4°C). The resulting plasma (supernatant) was CO:COLLECTION_SUMMARY collected and aliquoted into 1.5ml Eppendorf tubes and stored at -80°C. CO:SAMPLE_TYPE Blood (plasma) #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. Treatment began three days after tumor cell TR:TREATMENT_SUMMARY implantation (bilateral tumors in n=4 per group). Mice were euthanized17 days TR:TREATMENT_SUMMARY after tumor cell injection/14 days after the start of treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For plasma analysis, 20 µL of plasma was aliquoted into 1.5 mL Eppendorf tubes SP:SAMPLEPREP_SUMMARY followed by 480 µL cold MeOH. Samples were then vortexed for 30 minutes, SP:SAMPLEPREP_SUMMARY insoluble material was pelleted by centrifugation (18000g, 10 min) and SP:SAMPLEPREP_SUMMARY 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% water; 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 DS2-039-021 DS2-039-022 DS2-039-023 DS2-039-024 DS2-039-025 DS2-039-026 DS2-039-027 DS2-039-028 DS2-039-029 DS2-039-030 DS2-039-031 DS2-039-032 DS2-039-033 DS2-039-034 DS2-039-035 DS2-039-036 DS2-039-037 DS2-039-038 DS2-039-039 DS2-039-040 DS2-039-041 DS2-039-042 DS2-039-043 DS2-039-044 Factors factor:vehicle + low oleic acid diet | Sample source:plasma factor:vehicle + low oleic acid diet | Sample source:plasma factor:vehicle + low oleic acid diet | Sample source:plasma factor:vehicle + low oleic acid diet | Sample source:plasma factor:vehicle + low oleic acid diet | Sample source:plasma factor:vehicle + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:SCD inhibitor + low oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:Vehicle + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma factor:SCD inhibitor + high oleic acid diet | Sample source:plasma C16:0 FA 1068092096 1034615808 1228964992 1253437568 1186953216 1217445632 1183136128 1286904064 1255541632 1177618304 2703971328 2693680384 956410624 1106090880 862887808 1104197120 959632384 884266240 3057646336 3209257472 3224534528 3390637056 1469727744 1421308288 C18:0 FA 1342255616 1213252352 1170602112 1237283840 1221595904 1263201664 1555300992 1627966976 1633287424 1544106752 3082416128 3112625408 1153022592 1072986112 1105669120 1395418752 1035673856 1004901568 2828628224 2769453568 3884630272 4070535680 1621886976 1407111808 C18:1 FA 269712768 265890864 633812096 654936576 617892864 598859776 378287968 368021024 247274944 249309440 837813568 836100864 683874752 681507648 864111488 911480768 1174499968 1114871168 1930619392 1974487296 2243201024 2259309056 749385472 699758080 C18:2 FA 188701520 193494176 505038496 507853888 439967136 425279136 553066432 546902400 385467840 408732832 1560885248 1511747456 324066112 330119744 459018752 478663040 585638208 539410176 1520642304 1604210688 1704316544 1768251008 725970112 709615168 C16:3 FA 426581 387790 742807 738381 1068061 896930 551724 555041 533386 453119 2007510 1858369 544489 588556 451107 481066 1165827 1174663 1789772 1971823 3074910 3457319 1577394 1639579 α-C18:3 FA 14810705 14671789 52236408 50227328 40948252 40265720 45272836 43730476 28614712 29620930 102287040 96123552 27674342 29607942 21432696 22599046 29599728 26512366 113523736 117690800 89947304 92601816 53601780 52078764 γ-C18:3 FA 3768389 3794803 9168488 9510603 9033843 9214382 7988708 7475910 4746707 5410168 20311884 17872856 4590921 5585135 2708979 3603708 6356941 5616926 21529966 23349328 22710000 25222676 7232931 6882220 C16:1 FA 40010160 36550668 127035840 129316040 177500592 171313872 21502452 19260164 18810182 20370988 54418696 55882292 65398852 68940304 69539992 74272288 117946592 114671488 62523512 68913696 69566832 73019504 43838216 42952880 C16:2 FA 1436413 1156975 2547435 2254478 2916320 2966571 1426675 1291588 1632586 1763853 5790658 4999233 1674716 2151863 2314416 2457788 3660141 3406733 6037918 6374203 6055246 6357073 4183968 4129049 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Lipid Class Polarity m/z rt C16:0 FA FA 17MM- 255.232742 8.839 C18:0 FA FA 17MM- 283.264374 10.127 C18:1 FA FA 17MM- 281.248566 9.094 C18:2 FA FA 17MM- 279.232941 8.101 C16:3 FA FA 17MM- 249.186172 5.964 α-C18:3 FA FA 17MM- 277.217346 7.188 γ-C18:3 FA FA 17MM- 277.217438 7.318 C16:1 FA FA 17MM- 253.217117 7.727 C16:2 FA FA 17MM- 251.201523 6.741 METABOLITES_END #END