MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_negative_reverse-phase-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_positive_reverse-phase-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_negative_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_positive_reverse-phase-3_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/m_MTBLS14278_LC-MS_positive_reverse-phase-2_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_positive_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_positive_reverse-phase-3.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_negative_reverse-phase-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_positive_reverse-phase-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_negative_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/a_MTBLS14278_LC-MS_positive_reverse-phase-2.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278/s_MTBLS14278.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14278Metabolite assignments and heavy isotopologue detection for absolute quantitation against internal standards were determined against in-house standard libraries and KEGG database searches through MavenMetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phasePrepare a 5 min gradient UHPLC-MS positive ion method with the following chromatography conditions: Flow rate 0.45 mL/min, column temperature 45 °C, and sample compartment temperature 7 °C. Solvent gradient is as follows: 0–0.5 min 5% B, 0.5–1.1 min 5–95% B, 1.1–2.75 min hold at 95% B, 2.75–3 min 95–5% B, and 3–5 min hold at 5% B. Utilize a C18 column (method has been optimized for Kinetex C18 column) and a C18 guard column. Phases for positive mode should be supplemented with 0.1% (v/v) formic acid.Aggressive breast cancers secrete heme metabolites to alter macrophage immune suppression and function.Michelle WilliamsUniversity of Pittsburghconditioned mediumblood plasmatumortumor interstitial fluidsolventpooled samplemass spectrometry assayExtraction of metabolites was performed as follows: 40 μL of sample was aliquoted into 2mL deep well plates followed by an addition of 360 μL cold MeOH:MeCN:H2O (5:3:2, v:v:v) for metabolomics or pure methanol for lipidomics. Plates were then placed on a shaker at 4°C and plate shaker was set to 400 RPM for 30 minutes. Mus musculusnot applicableHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS14278Michelle Williams. University of Pittsburgh. 5051 Center Ave Pittsburgh PA, 15213. mmw178@pitt.edu.Thomas Hintelmann. University of Pittsburgh. 5051 Center Ave Pittsburgh PA, 15213. THH75@pitt.edu.Raw files were converted to .mzXML using RawConverter.TreatmentCell lineGenotypeMMW178@pitt.eduCells Lines: All cells were grown in a humidified chamber supplemented with 5% carbon dioxide and kept at 37°-Celsius. 66Cl-4 cells were grown in DMEM-high glucose (Corning: SH30243.01) supplemented with 1% penicillin-streptomycin (pen/strep, Fisher Scientific: 15140-122), 1% L-glutamine (Fisher Scientific: 25030-081), 1% non-essential amino acids (NEAA, Fisher Scientific: 11140-050), and 10% fetal calf serum (FCS, HyClone: SH30073.03). The human TNBC cell line BT549 was grown in RMPI supplemented with 1% pen/strep, 1% NEAA, 10% FBS, and 5 mg/mL insulin (Sigma: I6634). All human cell lines were authenticated by short tandem repeat analysis (Promega) at the University of Colorado Anschutz Medical Campus (CU AMC) Tissue Culture Core in 2019. Murine Experiment Samples: 200,000 66Cl-4 parental, shCnt, shHO.1, or shHO.2 cells were injected into the 4th inguinal mammary fatpads of 8-week old host female BALB/CJ or BALB/CJ RAG2 KO/IL2Rgc KO/SIRPa(NOD) mice. Tumor volume was monitored after tumors became palpable every other day using calipers ((volume = 1/2(width2 × height)). When applicable, animals were randomized and tumor volume matched using excel, and treatment was initiated when average tumor volume reached 80 mm3. 25 mg/kg SnMP (Cayman Chemical, 19071) or vehicle control (4.5% sodium phosphate in sterile saline) were delivered daily by oral gavage. 250 mg/mouse anti-PD-1 antibody (BioXCell, RMP1-14) or IgG control antibody (BioXCell, HRPN) were delivered by intraperitoneal injections every three days. Endpoint was either when tumor volume reached over 500 mm3 per group or 2 weeks treatment. At endpoint, tumors were either formalin fixed paraffin embedded (FFPE) for histological analysis. Tumors generated from knockdown cell lines were centrifuged to collect interstitial fluid using protocol from (https://doi.org/10.1016/j.stem.2023.11.001). TIF from SnMP treated tumors was extracted by mashing tumors onto a filter and collecting the liquid. Blood was collected using a cardiac puncture and placed into EDTA coated tubes (FisherScientific, NC9414041) that were centrifuged using protocol from (https://doi.org/10.1016/j.stem.2023.11.001) to collect plasma for mass spectrometry analysis of biliverdin and bilirubin by the CU AMC Mass Spectrometry Metabolomics Shared Resource.  Metabolomicsconditioned mediumpooled quality control sampleMetabolomicsMus musculusnot applicableuntargeted analysisThermo Scientific Orbitrap Exploris 120tumor interstitial fluidsolvent blankHomo sapiensbreast cancerpooled samplebilirubin IXalphaexperimental sampleThermo Scientific Vanquish UHPLC Systemblood plasmametastasistumorImmunotherapysolventheme catabolic processheme oxygenase 1tumor metabolismMacrophageconditioned mediumpooled quality control sampleMetabolomicsMus musculusnot applicableuntargeted analysisThermo Scientific Orbitrap Exploris 120solvent blanktumor interstitial fluidHomo sapiensbreast cancerpooled samplebilirubin IXalphaexperimental sampleThermo Scientific Vanquish UHPLC Systemblood plasmametastasistumorImmunotherapysolventheme catabolic processtumor metabolismheme oxygenase 1MacrophageAdd mass spectrometry settings to the gradient UHPLC-MS method: resolution 70,000, scan range 65–900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 × 106 ions, source voltage 4.0 kV, capillary temperature 320 °C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Optimal ESI or HESI gas and voltage parameters may be determined by observing signal intensity as settings are changed. biliverdinBilirubinfalseAggressive breast cancers secrete heme metabolites to alter macrophage immune suppression and functionThe heme catabolism pathway is often elevated in aggressive cancers; however, the impact of this pathway and some of its byproducts on the tumor microenvironment remain largely unknown. In human breast cancers, tumor expression of the heme catabolizing enzyme heme oxygenase-1 (HO-1/HMOX1) is positively associated with macrophage abundance. In mouse mammary tumors, knockdown of Hmox1 significantly decreased tumor growth and lung metastasis. Analysis of mammary tumor interstitial fluid compared to matching plasma revealed that the heme metabolite bilirubin was elevated intratumorally, which could be partially reversed via Hmox1 knockdown. Further investigation revealed that bilirubin nearly ablates macrophage engulfment of dead tumor cells and significantly increases macrophage T cell suppression. Mammary tumors harboring Hmox1 knockdown had a significant decrease in tumor growth rate and number of pro-metastatic CD206+ macrophages upon treatment with αPD-1. Depletion of intratumoral bilirubin levels impacts pro-tumor macrophage populations, particularly in combination with immunotherapy, demonstrating that heme catabolism and bilirubin act as immunomodulators in cancer.2026-04-242026-04-14MTBLS14278MTBLC16990MTBLC17033CHEBI:16990CHEBI:17033