{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002935","ANALYSIS_ID":"AN004814","VERSION":"1","CREATED_ON":"October 17, 2023, 1:19 pm"},

"PROJECT":{"PROJECT_TITLE":"Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth","PROJECT_TYPE":"LC-MS/MS","PROJECT_SUMMARY":"Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and human GBM patients identified the de-novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). Therefore, we hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine can be taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth, and enhanced radiation therapy in-vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.","INSTITUTE":"Northwestern University, Feinberg School of Medicine","DEPARTMENT":"Neurological Surgery","LABORATORY":"Jason Miska","LAST_NAME":"Miska","FIRST_NAME":"Jason","ADDRESS":"676 N St. Clair","EMAIL":"jason.miska@northwestern.edu","PHONE":"8478678201"},

"STUDY":{"STUDY_TITLE":"Title: Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth","STUDY_TYPE":"PBMC vs Tumor CD163+","STUDY_SUMMARY":"Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and human GBM patients identified the de-novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). Therefore, we hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine can be taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth, and enhanced radiation therapy in-vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.","INSTITUTE":"Northwestern University, Feinberg School of Medicine","DEPARTMENT":"Neurological Surgery","LABORATORY":"Jason Miska","LAST_NAME":"Miska","FIRST_NAME":"Jason","ADDRESS":"676 N St. Clair","EMAIL":"jason.miska@northwestern.edu","PHONE":"8478678201","NUM_GROUPS":"2","TOTAL_SUBJECTS":"5"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-13",
"Factors":{"Treatment":"PBMC"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-13"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-14",
"Factors":{"Treatment":"PBMC"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-14"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-15",
"Factors":{"Treatment":"PBMC"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-15"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-16",
"Factors":{"Treatment":"Tumor"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-16"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-17",
"Factors":{"Treatment":"Tumor"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-17"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20190812-18",
"Factors":{"Treatment":"Tumor"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20190812-18"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20200214-01",
"Factors":{"Treatment":"Tumor"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20200214-01"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20200214-02",
"Factors":{"Treatment":"Tumor"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20200214-02"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20200214-03",
"Factors":{"Treatment":"PBMC"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20200214-03"}
},
{
"Subject ID":"-",
"Sample ID":"Les-Jas-20200214-04",
"Factors":{"Treatment":"PBMC"},
"Additional sample data":{"RAW_FILE_NAME":"Les-Jas-20200214-04"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Magnetic bead isolation of cells To isolate specific populations of cells, single-cell suspensions as isolated above are preblocked with anti-CD16/32 for 15 min at 4°C. We then used the biotinylated anti-CD163 (all from Thermo Fisher Scientific) to label murine myeloid cells. Next, the cells were washed and then incubated with anti-biotin magnetic beads (Miltenyi Biotec) before performing manual positive selection using MS columns (Miltenyi Biotec). Purified cells were analyzed for all downstream metabolic analyses.","SAMPLE_TYPE":"Brain"},

"TREATMENT":{"TREATMENT_SUMMARY":"Fresh cells isolated from PBMC or TIL of GBM patients"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Isolated TAMC samples were dried using a SpeedVac. Acetonitrile (50%) was added to the tube for reconstitution following overtaxing for 30 s. Sample solution was then centrifuged for 15 min at 20,000g and 4°C. Supernatant was collected for LC-MS analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Samples were analyzed by high-performance LC (HPLC) and high-resolution MS and MS/MS (HPLC-MS/MS). The system consists of Thermo Q Exactive with an electrospray source and an UltiMate3000 (Thermo Fisher Scientific) series HPLC consisting of a binary pump, degasser, and autosampler outfitted with an XBridge Amide column (Waters; dimensions of 4.6 mm by 100 mm and a 3.5-μm particle size). The mobile phase A contained 95% water/5% acetonitrile (v/v), 20 mM ammonium hydroxide, and 20 mM ammonium acetate (pH 9.0); phase B was 100% acetonitrile. The gradient was performed as follows: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1 to 18 min, 75% A; and 18 to 25 min, 15% A with a flow rate of 400 µl/min.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Water''s Xbridge amide (100 x 3mm, 3.5 um)","SOLVENT_A":"95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate (pH 9.0)","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1 to 18 min, 75% A; and 18 to 25 min, 15% A","FLOW_RATE":"400 μl/min","COLUMN_TEMPERATURE":"-"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The capillary of the electrospray ionization source was set to 275°C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units, and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, a mass/charge ratio (m/z) scan range from 70 to 850 was chosen and MS1 data were collected at a resolution of 70,000. The automatic gain control target was set at 1 × 106, and the maximum injection time was 200 ms. The top five precursor ions were subsequently fragmented, in a data-dependent manner, using the higher-energy collisional dissociation cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500."},

"MS_METABOLITE_DATA":{
"Units":"Peak Area",

"Data":[{"Metabolite":"1,3-bisphospho-D-glycerate","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"2-(Formamido)-N1-(5-phospho-D-ribosyl)acetamidine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"2,3-bisphospho-D-glycerate","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"2190732.466","Les-Jas-20200214-02":"143113.0952","Les-Jas-20200214-03":"177631.9904","Les-Jas-20200214-04":"299048.3901"},{"Metabolite":"2/3-phosphoglycerate","Les-Jas-20190812-13":"46141.81004","Les-Jas-20190812-14":"53535.47435","Les-Jas-20190812-15":"54367.90792","Les-Jas-20190812-16":"49863.91701","Les-Jas-20190812-17":"520676.4536","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"11958211.65","Les-Jas-20200214-02":"2749000.17","Les-Jas-20200214-03":"3458662.387","Les-Jas-20200214-04":"3830196.175"},{"Metabolite":"2-HG","Les-Jas-20190812-13":"3371511.187","Les-Jas-20190812-14":"2002299.932","Les-Jas-20190812-15":"1322888.801","Les-Jas-20190812-16":"1435284.933","Les-Jas-20190812-17":"1056283.271","Les-Jas-20190812-18":"1694115.509","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"3,4-dihydroxymandelic acid","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"25016.94174","Les-Jas-20190812-17":"13712.69084","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"3,4-dihydroxyphenylpropanoate","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"3-hydroxypropanoic acid","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"60471344.28","Les-Jas-20200214-02":"29337300.69","Les-Jas-20200214-03":"24865805.21","Les-Jas-20200214-04":"71060908.42"},{"Metabolite":"3-methoxytyramine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"23311.53874","Les-Jas-20200214-01":"34650.32982","Les-Jas-20200214-02":"35229.04505","Les-Jas-20200214-03":"27325.00029","Les-Jas-20200214-04":"20799.93014"},{"Metabolite":"3-phosphohydroxypyruvic acid","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"3-phospho-serine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"52236.68162","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"25896.77836","Les-Jas-20200214-04":"17126.78827"},{"Metabolite":"3-ureidopropionic acid","Les-Jas-20190812-13":"16572.0674","Les-Jas-20190812-14":"11194.80957","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"15828.98507","Les-Jas-20200214-02":"34393.84684","Les-Jas-20200214-03":"10701.85039","Les-Jas-20200214-04":"13704.73516"},{"Metabolite":"4-phosphopantothenate","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"5,6-dihydroxyindole","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"17277.08803","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"14825.60444"},{"Metabolite":"5-methylcytosine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"429750.4434","Les-Jas-20200214-02":"112065.4204","Les-Jas-20200214-03":"294842.6677","Les-Jas-20200214-04":"115919.0531"},{"Metabolite":"5-methyl-THF","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"5''-methylthioadenosine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"38228.52188","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"1474186.72","Les-Jas-20200214-03":"904925.5263","Les-Jas-20200214-04":"1204594.402"},{"Metabolite":"6-aminocaproic acid","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"6-O-methylguanine","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"6-phosphogluconic acid","Les-Jas-20190812-13":"67173.05724","Les-Jas-20190812-14":"41381.73941","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"11961.94372","Les-Jas-20190812-18":"25886.32792","Les-Jas-20200214-01":"813720.0403","Les-Jas-20200214-02":"352696.007","Les-Jas-20200214-03":"177796.4493","Les-Jas-20200214-04":"110835.5411"},{"Metabolite":"6-phosphoglucono-D-lactone","Les-Jas-20190812-13":"0","Les-Jas-20190812-14":"0","Les-Jas-20190812-15":"0","Les-Jas-20190812-16":"0","Les-Jas-20190812-17":"0","Les-Jas-20190812-18":"0","Les-Jas-20200214-01":"0","Les-Jas-20200214-02":"0","Les-Jas-20200214-03":"0","Les-Jas-20200214-04":"0"},{"Metabolite":"acetoacetic 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2/3-phosphate"},{"metabolite_name":"glycine"},{"metabolite_name":"glycylglycine"},{"metabolite_name":"GMP"},{"metabolite_name":"GTP"},{"metabolite_name":"guanidinoacetate"},{"metabolite_name":"guanine"},{"metabolite_name":"guanosine"},{"metabolite_name":"gulonic-lactone"},{"metabolite_name":"histamine"},{"metabolite_name":"histidine"},{"metabolite_name":"homocysteine"},{"metabolite_name":"homoserine / theronine"},{"metabolite_name":"homovanillic acid"},{"metabolite_name":"hydroxyphenyllactic acid"},{"metabolite_name":"hypoxanthine"},{"metabolite_name":"IDA"},{"metabolite_name":"IDP"},{"metabolite_name":"imidazole-4-acetate"},{"metabolite_name":"IMP"},{"metabolite_name":"inosine"},{"metabolite_name":"itaconic acid"},{"metabolite_name":"kynurenine"},{"metabolite_name":"lactic acid"},{"metabolite_name":"L-arginino-succinate"},{"metabolite_name":"leucine / leucine(iso)"},{"metabolite_name":"levulinic acid"},{"metabolite_name":"L-NMMA"},{"metabolite_name":"lysine"},{"metabolite_name":"maleic acid"},{"metabolite_name":"malic acid"},{"metabolite_name":"methionine"},{"metabolite_name":"methylglyoxal"},{"metabolite_name":"methylmalonic acid"},{"metabolite_name":"mevalonic acid"},{"metabolite_name":"N1/N8-acetylspermidine"},{"metabolite_name":"N-acetylaspartic acid"},{"metabolite_name":"N-acetylaspartylglutamic acid"},{"metabolite_name":"N-acetyl-b-alanine"},{"metabolite_name":"N-acetyl-DL-alanine"},{"metabolite_name":"N-acetyl-dopamine"},{"metabolite_name":"N-acetylglucosamine 1/6-phosphate"},{"metabolite_name":"NAD+"},{"metabolite_name":"NADH"},{"metabolite_name":"NADP+"},{"metabolite_name":"NADPH"},{"metabolite_name":"N-carbamoyl-L-aspartate"},{"metabolite_name":"nicotinamide"},{"metabolite_name":"N-methyltryptamine"},{"metabolite_name":"NMN"},{"metabolite_name":"norepinephrine / pyridoxine"},{"metabolite_name":"ornithine"},{"metabolite_name":"orotic acid"},{"metabolite_name":"orotidylic acid"},{"metabolite_name":"oxalacetic acid"},{"metabolite_name":"pantothenic acid"},{"metabolite_name":"phenethylamine"},{"metabolite_name":"phenylalanine"},{"metabolite_name":"phosphocreatine"},{"metabolite_name":"phosphoenolpyruvate"},{"metabolite_name":"phosphorylcholine+"},{"metabolite_name":"proline"},{"metabolite_name":"PRPP"},{"metabolite_name":"pseudouridine"},{"metabolite_name":"putrescine"},{"metabolite_name":"pyridoxine"},{"metabolite_name":"pyruvic acid"},{"metabolite_name":"S-adenosyl-L-homocysteine"},{"metabolite_name":"S-adenosyl-L-methionine+"},{"metabolite_name":"S-adenosylmethioninamine+"},{"metabolite_name":"serine"},{"metabolite_name":"serotonin"},{"metabolite_name":"spermidine"},{"metabolite_name":"spermine"},{"metabolite_name":"succinic acid"},{"metabolite_name":"taurine"},{"metabolite_name":"thiamine+"},{"metabolite_name":"thymidine"},{"metabolite_name":"thymine"},{"metabolite_name":"tryptamine"},{"metabolite_name":"tryptophan"},{"metabolite_name":"tyramine"},{"metabolite_name":"tyrosine"},{"metabolite_name":"UDP"},{"metabolite_name":"UDP-D-glucose"},{"metabolite_name":"UDP-N-acetyl-galactosamine/UDP-N-acetyl-glucosamine"},{"metabolite_name":"UMP"},{"metabolite_name":"uracil"},{"metabolite_name":"urea"},{"metabolite_name":"uric acid"},{"metabolite_name":"uridine"},{"metabolite_name":"UTP"},{"metabolite_name":"valine"},{"metabolite_name":"xanthine"},{"metabolite_name":"XMP"},{"metabolite_name":"y-glutamylcysteine"}]
}

}