#METABOLOMICS WORKBENCH ramkhattri_20220609_131323 DATATRACK_ID:3301 STUDY_ID:ST002207 ANALYSIS_ID:AN003610 PROJECT_ID:PR001410 VERSION 1 CREATED_ON July 6, 2022, 9:13 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis to assess response to immunotherapy for malignant brain PR:PROJECT_TITLE tumors: Part 1 PR:PROJECT_TYPE Study of the urine and serum in mice treated with DC vaccine via 1H NMR PR:PROJECT_SUMMARY The objective of this project was to identify a peripheral metabolomic profile PR:PROJECT_SUMMARY to serve as a biomarker of response to immunotherapy for the treatment of PR:PROJECT_SUMMARY malignant brain tumors. PR:INSTITUTE University of Florida PR:DEPARTMENT Neurosurgery Department, Medical college, University of Florida PR:LAST_NAME Khattri PR:FIRST_NAME Ram PR:ADDRESS 1200 Newell Dr., ARB 240, Gainesville, FL, 32611, USA PR:EMAIL rbk11@ufl.edu PR:PHONE 3307856045 PR:FUNDING_SOURCE This work was partially funded by the Florida Center for Brain Tumor Research PR:FUNDING_SOURCE (FCBTR), and by generous benefactors to the University of Florida, Peter and PR:FUNDING_SOURCE Angela Dziegielewski, who established the Eilzabeth Dziegielewski Glioblastoma PR:FUNDING_SOURCE Research Fund, and Rosalinde Wolfe, who established the Greg Wolfe Brain Tumor PR:FUNDING_SOURCE Research Fund. RK and MM were supported by funding from National Institutes of PR:FUNDING_SOURCE Health (U24-DK097209 and 5U2C-DK119889). All NMR portion of this study was PR:FUNDING_SOURCE performed in McKnight Brain Institute at National High Magnetic Field PR:FUNDING_SOURCE Laboratory’s Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) PR:FUNDING_SOURCE Facility, which is funded by National Science Foundation Cooperative Agreement PR:FUNDING_SOURCE No. DMR-1644779 and the State of Florida. PR:PROJECT_COMMENTS Study of the urine and serum in mice treated with DC vaccine via 1H NMR PR:PUBLICATIONS Metabolomics journal (submitted) PR:CONTRIBUTORS Farhad Dastmalchi, Ram B. Khattri, Marc A. McLeod, Kaitlyn Melnick, Loic P. PR:CONTRIBUTORS Deleyrolle, Yusuf Mehkri, Aida Karachi, Paul Kubilis, Shu Wang, Duane A. PR:CONTRIBUTORS Mitchell, Matthew E. Merritt, Maryam Rahman #STUDY ST:STUDY_TITLE Metabolomic analysis to assess response to immunotherapy for malignant brain ST:STUDY_TITLE tumors: Part 1 ST:STUDY_SUMMARY An effective immune response in patients with cancer treated with immunotherapy ST:STUDY_SUMMARY includes dendritic cell (DC) activation and migration followed by stimulation of ST:STUDY_SUMMARY CD8 and CD4 T cells. This then leads to the activation, proliferation and ST:STUDY_SUMMARY further activation of other immune cell populations including NK cells or ST:STUDY_SUMMARY immunosuppressive populations such as Tregs and myeloid derived suppressor cells ST:STUDY_SUMMARY (MDSCs). These studies were carried out utilizing murine brain tumor models ST:STUDY_SUMMARY treated with an RNA DC vaccine platform. We hypothesized that metabolomic ST:STUDY_SUMMARY analyses of urines would be sensitive to the action of this diverse set of ST:STUDY_SUMMARY immune cells. The objective of this study was to evaluate the feasibility of ST:STUDY_SUMMARY using metabolomics to follow immune responses after immunotherapy. We chose NMR ST:STUDY_SUMMARY as our analytical technique of choice, as it has many favorable qualities that ST:STUDY_SUMMARY make it ideal for analyses of urine. ST:INSTITUTE University of Florida ST:DEPARTMENT Neurosurgery ST:LABORATORY Rm 042 ST:LAST_NAME Khattri ST:FIRST_NAME Ram ST:ADDRESS 1864 Stadium RD, ST:EMAIL rbk11@ufl.edu ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 15 ST:NUM_MALES NA ST:NUM_FEMALES NA ST:STUDY_COMMENTS Metabolomic profiling of urine samples ST:PUBLICATIONS Metabolomics journal (submitted) ST:STUDY_TYPE Cancer surrogate biomarker discovery ST:PHONE 330-785-6045 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Bar Harbor, ME) SU:ANIMAL_HOUSING Housed in a temperature of 22 oC SU:ANIMAL_LIGHT_CYCLE 12-hour light/12-hour dark SU:ANIMAL_WATER free access to food and water (3-5 animals per cage). #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 - Maryam_1st-set_Urine_Control-1 Group:Control RAW_FILE_NAME=Maryam_1st-set_Urine_Control-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_Control-2 Group:Control RAW_FILE_NAME=Maryam_1st-set_Urine_Control-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_Control-3 Group:Control RAW_FILE_NAME=Maryam_1st-set_Urine_Control-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_Control-4 Group:Control RAW_FILE_NAME=Maryam_1st-set_Urine_Control-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_Control-5 Group:Control RAW_FILE_NAME=Maryam_1st-set_Urine_Control-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_24hrs-1 Group:24 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_24hrs-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_24hrs-2 Group:24 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_24hrs-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_24hrs-3 Group:24 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_24hrs-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_24hrs-4 Group:24 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_24hrs-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_24hrs-5 Group:24 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_24hrs-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_48hrs-1 Group:48 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_48hrs-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_48hrs-2 Group:48 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_48hrs-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_48hrs-3 Group:48 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_48hrs-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_48hrs-4 Group:48 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_48hrs-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_1st-set_Urine_48hrs-5 Group:48 hrs RAW_FILE_NAME=Maryam_1st-set_Urine_48hrs-5.raw #COLLECTION CO:COLLECTION_SUMMARY Intakt urine samples were collected from C57/Bl6 mice. Urine is collected from a CO:COLLECTION_SUMMARY clean surface after urination. CO:SAMPLE_TYPE Urine CO:COLLECTION_METHOD Naïve C57/BL6 mice received antigen-specific T cells through intravenous (IV) CO:COLLECTION_METHOD and the DC vaccine injection intradermally on the same day. Both CO:COLLECTION_METHOD antigen-specific T cells and DC vaccine were administered once. Urine or serum CO:COLLECTION_METHOD samples were collected after DC vaccination at several timepoints. Finally, CO:COLLECTION_METHOD animals were euthanized when they reached the endpoints CO:COLLECTION_LOCATION University of Florida, Neurosurgery Department, Medical college, University of CO:COLLECTION_LOCATION Florida CO:COLLECTION_FREQUENCY Pre-vaccination, 24 hrs and 48 hrs of post DC vaccination. CO:COLLECTION_DURATION ~30 minutes CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS cryovials CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY Dendritic cell vaccine and anti-PD-1 immunotherapy. Bone marrow (BM) was TR:TREATMENT_SUMMARY harvested from the long bones and sternum of euthanized animals and the red TR:TREATMENT_SUMMARY blood cells (RBCs) were lysed. Then myeloid-derived cells were cultured in DC TR:TREATMENT_SUMMARY complete media including granulocyte macrophage colony stimulating factor TR:TREATMENT_SUMMARY (GM-CSF) and interleukin 4. Cells were cultured in six well plates for six days. TR:TREATMENT_SUMMARY On day seven, cells were re-plated in 60mm dishes. DCs were then electroporated TR:TREATMENT_SUMMARY with OVA-mRNA at day eight. On day nine, DCs were collected in phosphate TR:TREATMENT_SUMMARY buffered saline (PBS) for administration. DC vaccines were delivered once via TR:TREATMENT_SUMMARY intradermal injection in the inguinal area. Naïve C57/BL6 mice received TR:TREATMENT_SUMMARY antigen-specific T cells through intravenous (IV) and the DC vaccine injection TR:TREATMENT_SUMMARY intradermally on the same day. Both antigen-specific T cells and DC vaccine were TR:TREATMENT_SUMMARY administered once. Urine or serum samples were collected after DC vaccination at TR:TREATMENT_SUMMARY several timepoints. Finally, animals were euthanized when they reached the TR:TREATMENT_SUMMARY endpoints. TR:ANIMAL_ANESTHESIA isoflurane TR:ANIMAL_FASTING non-fasted TR:ANIMAL_ENDP_EUTHANASIA Euthanasia was carried out by thoracotomy followed by cervical dislocation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY No filtration was performed for urine samples. The urine samples were SP:SAMPLEPREP_SUMMARY centrifuged at 4 oC, with 13.2 K rpm speed before mixing it with an internal SP:SAMPLEPREP_SUMMARY reference and phosphate buffer system. SP:SAMPLEPREP_PROTOCOL_FILENAME 1. DC-Vaccine-treated-NMR urine Procedures SP:PROCESSING_METHOD None SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD None SP:SAMPLE_RESUSPENSION In 500 microliter of 150 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 10 mM TSP SP:SAMPLE_RESUSPENSION and 0.2% sodium azide for aqueous phase samples. SP:SAMPLE_SPIKING 10 mM of TSP for urine samples #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME McKnight Brain Institute AN:OPERATOR_NAME Ram Khattri AN:DETECTOR_TYPE Bruker 600 MHz AN:SOFTWARE_VERSION Topspin AN:ACQUISITION_DATE 3/9/2016 AN:ACQUISITION_PARAMETERS_FILE 1. DC-Vaccine-treated-NMR urine Procedures AN:PROCESSING_PARAMETERS_FILE 1. DC-Vaccine-treated-NMR urine Procedures AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker 600 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 10 mM TSP NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 5 mm CPTXI 1H/D-13C/15N Z-GRD Z44866/0026 NM:NMR_SOLVENT Phosphate buffer (pH 7.2) + 2 mM EDTA + 10 mM TPS + 0.2% of sodium azide in NM:NMR_SOLVENT deuterated environment NM:NMR_TUBE_SIZE 5 mm O.D. NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 256 NM:OFFSET_FREQUENCY 4.77 ppm NM:CHEMICAL_SHIFT_REF_CPD TPS NM:TEMPERATURE 300.2 oK NM:NUMBER_OF_SCANS 256 NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 2.2719 s NM:RELAXATION_DELAY 3 s NM:SPECTRAL_WIDTH 7211.5 Hz NM:NUM_DATA_POINTS_ACQUIRED 16384 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.22 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Spline NM:CHEMICAL_SHIFT_REF_STD 0 ppm for TPS NM:BINNED_INCREMENT 0.001 ppm NM:BINNED_DATA_EXCLUDED_RANGE >9.5 ppm and < 0.5 ppm regions NM:NMR_RESULTS_FILE ST002207_AN003610_Results.txt UNITS:Peak intensity #END