{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002530","ANALYSIS_ID":"AN004164","VERSION":"1","CREATED_ON":"March 28, 2023, 10:13 am"},

"PROJECT":{"PROJECT_TITLE":"Pancreatic tumors activate arginine biosynthesis to adapt to myeloid-driven amino acid stress","PROJECT_SUMMARY":"Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs to maintain survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors (Sullivan et al., 2019a). Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling study of PDAC metabolism under physiological nutrition. We show that PDAC cells cultured in TIFM, compared to standard laboratory models, adopt a cellular state more similar to PDAC cells in tumors. Further, using the TIFM model we identified arginine biosynthesis as a metabolic adaptation PDAC cells engage to cope with microenvironmental arginine starvation driven by myeloid cells in PDAC tumors. Altogether, these data show that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity and enable the discovery of novel cancer metabolic phenotypes.","INSTITUTE":"University of Chicago","LAST_NAME":"Apiz Saab","FIRST_NAME":"Juan","ADDRESS":"929 E. 57th St.","EMAIL":"japizsaab@uchicago.edu","PHONE":"7738346506"},

"STUDY":{"STUDY_TITLE":"LCMS analysis of amino acid levels in PDAC interstitial fluid samples upon ass1 cancer cell knock out","STUDY_SUMMARY":"We used CRISPR-Cas9 to knockout (KO) Ass1 in mPDAC cells. We tested if inhibiting arginine de novo synthesis would impair PDAC tumor growth. To test this, we generated orthotopic PDAC tumors with mPDAC-Ass1 KO cells and control cells where Ass1 was re-expressed (Ass1KO; mASS1).","INSTITUTE":"University of Chicago","LAST_NAME":"Apiz Saab","FIRST_NAME":"Juan","ADDRESS":"929 E. 57th St.","EMAIL":"japizsaab@uchicago.edu","PHONE":"7738346506"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"20210804_01",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-01-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_02",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-02-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_05",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-05-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_08",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-08-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_10",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-10-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_11",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-11-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_13",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-13-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_17",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-17-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_19",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-19-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_20",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-20-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_22",
"Factors":{"Sample type":"ASS1KO"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-22-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_03",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-03-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_04",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-04-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_06",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-06-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_07",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-07-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_09",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-09-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_12",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-12-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_14",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-14-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_15",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-15-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_16",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-16-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_18",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-18-.raw"}
},
{
"Subject ID":"-",
"Sample ID":"20210804_21",
"Factors":{"Sample type":"ASS1KO;mASS1"},
"Additional sample data":{"RAW_FILE_NAME":"Mui-Ale-20210804-21-.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Tumors were rapidly dissected after euthanizing animals. Tumors were weighed and rinsed in blood bank saline solution (150 mM NaCl) and blotted on filter paper (VWR, Radnor, PA, 28298–020). The process of dissection and tumor preparation took < 3min. Tumors were cut in half and put onto 20µm nylon mesh filters (Spectrum Labs, Waltham, MA, 148134) on top of 50 mL conical tubes, and centrifuged for 10min. at 4°C at 400xg. IF was then collected, snap-frozen in liquid nitrogen and stored at -80°C until further analysis.","SAMPLE_TYPE":"Interstitial Fluid"},

"TREATMENT":{"TREATMENT_SUMMARY":"Orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after induction interstitial fluid and tumors were collected."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"polar metabolites were extracted from 5µL IF samples using 45µL 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid extraction mix into which a mixture of isotopically labeled amino acids of known concentrations (Cambridge Isotope Laboratories, MSK-A2-1.2) was added. Samples were vortexed for 10 min, and centrifuged at maximum speed for 10 min. 30 μL of each extract was removed and dried under nitrogen gas and stored −80°C."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"HPLC","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters XBridge Amide (100 x 4.6mm, 3.5um)","SOLVENT_A":"95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate, pH = 9.0","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"0 min, 15% A; 2.5 min, 64% A; 12.4 min, 40% A; 12.5 min, 30% A; 12.5-14 min, 30% A; 14-21 min, 15% A","FLOW_RATE":"150 μL/min","COLUMN_TEMPERATURE":"25"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The targeted ions were subsequently fragmented, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Besides matching m/z, target metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific)."},

"MS_METABOLITE_DATA":{
"Units":"Peak area (m/z)",

"Data":[{"Metabolite":"alanine","20210804_01":"767374489","20210804_02":"777249191","20210804_05":"756971607","20210804_08":"437041064","20210804_10":"481277536","20210804_11":"890966100","20210804_13":"925359610","20210804_17":"679885821","20210804_19":"339932299","20210804_20":"896799432","20210804_22":"979407383","20210804_03":"917387748","20210804_04":"592429379","20210804_06":"271084133","20210804_07":"639805345","20210804_09":"582879208","20210804_12":"627972132","20210804_14":"562873140","20210804_15":"830653814","20210804_16":"650058827","20210804_18":"565222900","20210804_21":"827250875"},{"Metabolite":"alanine_13C3_15N","20210804_01":"8227648","20210804_02":"7200302","20210804_05":"6812217","20210804_08":"7305998","20210804_10":"7467993","20210804_11":"6176263","20210804_13":"8194685","20210804_17":"7773241","20210804_19":"5315795","20210804_20":"7416957","20210804_22":"7049154","20210804_03":"7464871","20210804_04":"7182348","20210804_06":"6064572","20210804_07":"7860594","20210804_09":"8017385","20210804_12":"8647310","20210804_14":"8407094","20210804_15":"7201381","20210804_16":"7695638","20210804_18":"7233349","20210804_21":"7050010"},{"Metabolite":"arginine","20210804_01":"81989938","20210804_02":"66850506","20210804_05":"28865877","20210804_08":"110069587","20210804_10":"128783026","20210804_11":"24994825","20210804_13":"66688818","20210804_17":"51331729","20210804_19":"68021772","20210804_20":"17865837","20210804_22":"39521174","20210804_03":"12937081","20210804_04":"140817272","20210804_06":"63115237","20210804_07":"23921488","20210804_09":"179588936","20210804_12":"20145457","20210804_14":"243521867","20210804_15":"17488566","20210804_16":"211092965","20210804_18":"70433054","20210804_21":"45587325"},{"Metabolite":"arginine_13C6_15N4","20210804_01":"59641608","20210804_02":"49500616","20210804_05":"47269338","20210804_08":"46308858","20210804_10":"52635004","20210804_11":"44727387","20210804_13":"68037628","20210804_17":"50178754","20210804_19":"31196140","20210804_20":"60300655","20210804_22":"73640739","20210804_03":"60093131","20210804_04":"49649465","20210804_06":"35258506","20210804_07":"66899604","20210804_09":"66686739","20210804_12":"66422093","20210804_14":"68769173","20210804_15":"64579238","20210804_16":"68148217","20210804_18":"57728209","20210804_21":"68016636"},{"Metabolite":"asparagine","20210804_01":"158356777","20210804_02":"158793383","20210804_05":"160737583","20210804_08":"117051994","20210804_10":"111206422","20210804_11":"159868501","20210804_13":"243095881","20210804_17":"126495741","20210804_19":"67150072","20210804_20":"166903355","20210804_22":"194740389","20210804_03":"176357268","20210804_04":"98842701","20210804_06":"51675369","20210804_07":"129591794","20210804_09":"123829444","20210804_12":"131294086","20210804_14":"121613953","20210804_15":"154801571","20210804_16":"159302848","20210804_18":"127607888","20210804_21":"168701764"},{"Metabolite":"aspartic 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