{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002489","ANALYSIS_ID":"AN004063","VERSION":"1","CREATED_ON":"February 21, 2023, 12:18 pm"},

"PROJECT":{"PROJECT_TITLE":"Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit pancreatic cancer growth and metastasis","PROJECT_SUMMARY":"In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL2Rβ and IL2Rγ and decreased ILR2α expression. The bispecific aPD1-IL2v is a PD-1-targeted IL-2 variant (IL2v) immunocytokine with engineered IL-2 cis-targeted to PD-1 and abolished IL2Rα binding, which enhances tumor-antigen specific T cell activation while reducing regulatory T cell (Treg) suppression. Using aPD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival along with profound increase in tumor-infiltrating polyfunctional CD8+ T cell subsets with a transcriptionally and metabolically active phenotype, and preferential activation of antigen-specific CD8+ T cells. In combination with single dose RT, aPD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that aPD1-IL2v leads to profound local and distant response in PDAC.","INSTITUTE":"University of Colorado Denver","LABORATORY":"Lab of Angelo D'Alessandro in collaboration with lab of Sana Karam","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"STUDY":{"STUDY_TITLE":"Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit pancreatic cancer growth and metastasis","STUDY_SUMMARY":"Mice were orthotopically implanted with PK5L1940 cells then radiation therapy (8 Gy) administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. T-cells from spleen were obtained through sorting, cultured in the presence of 13C6 glucose for 6h, and glucose fluxes profiled via mass spectrometry-based metabolomics.","INSTITUTE":"University of Colorado Denver","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-1",
"Factors":{"factor":"unx"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-1_r6-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-2",
"Factors":{"factor":"unx"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-2_r4-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-3",
"Factors":{"factor":"unx"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-3_r3-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-4",
"Factors":{"factor":"RT"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-4_r8-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-5",
"Factors":{"factor":"RT"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-5_r12-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-6",
"Factors":{"factor":"RT"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-6_r11-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-7",
"Factors":{"factor":"IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-7_r14-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-8",
"Factors":{"factor":"IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-8_r1-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-9",
"Factors":{"factor":"IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-9_r5-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-10",
"Factors":{"factor":"RT+IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-10_r2-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-11",
"Factors":{"factor":"RT+IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-11_r9-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-12",
"Factors":{"factor":"RT+IL2v"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-12_r15-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-13",
"Factors":{"factor":"aCD25"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-13_r7-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-14",
"Factors":{"factor":"aCD25"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-14_r13-"}
},
{
"Subject ID":"-",
"Sample ID":"AG-23-TC-15",
"Factors":{"factor":"aCD25"},
"Additional sample data":{"RAW_FILE_NAME":"AG-23-TC-15_r10-"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"C57BL/6 mice were orthotopically implanted with PK5L1940 cells. 8 Gy RT was administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. Spleens and lymph nodes of C57BL/6 mice were harvested 17 days post implantation and homogenized to a single cell suspension by mashing through a 70 uM cells strainer. For spleen processing, and the erythrocytes were lysed with ACK (ammonium-chloride-potassium) lysis buffer for 5 min at RT. CD8 T cells were sorted with a CD8-negative selection Stemcell isolation kit following manufacturer instructions.","SAMPLE_TYPE":"T-cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"CD8+ T cells were cultured for 6h in the presence of 25 mM U-13C-glucose in glucose-free RPMI. Cells were washed in PBS at 1200RPM, 4degC for 10 minutes and pellets were flash frozen."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolites from frozen T-cell pellets were extracted with cold 5:3:2 MeOH:acetonitrile:water at a concentration of 4e6 cells/ml. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.","PROCESSING_STORAGE_CONDITIONS":"4℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Negative C18","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um","SOLVENT_A":"95% water 5% acetonitrile 1 mM ammonium acetate","SOLVENT_B":"95% acetonitrile 5% water 1 mM ammonium acetate","FLOW_GRADIENT":"0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B","FLOW_RATE":"450 uL/min","COLUMN_TEMPERATURE":"45","SAMPLE_INJECTION":"10 uL"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

"Data":[{"Metabolite":"Glucose_M+0","AG-23-TC-1":"1989593","AG-23-TC-2":"572482.3","AG-23-TC-3":"1483604","AG-23-TC-4":"2388277","AG-23-TC-5":"1042860","AG-23-TC-6":"1753610","AG-23-TC-7":"1873150","AG-23-TC-8":"2008127","AG-23-TC-9":"2005074","AG-23-TC-10":"932287.3","AG-23-TC-11":"1873801","AG-23-TC-12":"2422508","AG-23-TC-13":"1578506","AG-23-TC-14":"1490044","AG-23-TC-15":"1514221"},{"Metabolite":"Glucose_M+6","AG-23-TC-1":"2.20E+07","AG-23-TC-2":"9776315","AG-23-TC-3":"2.08E+07","AG-23-TC-4":"2.34E+07","AG-23-TC-5":"1.37E+07","AG-23-TC-6":"1.60E+07","AG-23-TC-7":"2.02E+07","AG-23-TC-8":"2.00E+07","AG-23-TC-9":"2.21E+07","AG-23-TC-10":"1.54E+07","AG-23-TC-11":"2.14E+07","AG-23-TC-12":"1.96E+07","AG-23-TC-13":"2.14E+07","AG-23-TC-14":"1.94E+07","AG-23-TC-15":"1.90E+07"},{"Metabolite":"Hexose-6-phosphate_M+0","AG-23-TC-1":"11817.44","AG-23-TC-2":"3017.994","AG-23-TC-3":"10235.61","AG-23-TC-4":"6972.78","AG-23-TC-5":"6931.506","AG-23-TC-6":"13089.78","AG-23-TC-7":"13132.64","AG-23-TC-8":"6147.406","AG-23-TC-9":"10689.89","AG-23-TC-10":"6541.762","AG-23-TC-11":"10195.31","AG-23-TC-12":"9501.78","AG-23-TC-13":"10402.26","AG-23-TC-14":"6550.748","AG-23-TC-15":"4972.391"},{"Metabolite":"Hexose-6-phosphate_M+6","AG-23-TC-1":"18339.7","AG-23-TC-2":"6564.603","AG-23-TC-3":"14239.47","AG-23-TC-4":"16004.5","AG-23-TC-5":"42505.54","AG-23-TC-6":"18478.29","AG-23-TC-7":"15194.23","AG-23-TC-8":"10376.95","AG-23-TC-9":"30355.51","AG-23-TC-10":"18428.38","AG-23-TC-11":"19328","AG-23-TC-12":"21523.23","AG-23-TC-13":"10927.41","AG-23-TC-14":"19819.15","AG-23-TC-15":"14362.22"},{"Metabolite":"D-Fructose 1-6-bisphosphate_M+0","AG-23-TC-1":"29005.78","AG-23-TC-2":"42160.6","AG-23-TC-3":"50077.98","AG-23-TC-4":"20851.47","AG-23-TC-5":"22811.56","AG-23-TC-6":"32691.87","AG-23-TC-7":"29122.48","AG-23-TC-8":"51798.53","AG-23-TC-9":"34634.48","AG-23-TC-10":"88245.56","AG-23-TC-11":"30589.52","AG-23-TC-12":"16783.07","AG-23-TC-13":"51781.52","AG-23-TC-14":"26417.01","AG-23-TC-15":"50672.77"},{"Metabolite":"D-Fructose 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}