#METABOLOMICS WORKBENCH ramkhattri_20220609_140630 DATATRACK_ID:3303 STUDY_ID:ST002209 ANALYSIS_ID:AN003612 PROJECT_ID:PR001410 VERSION 1 CREATED_ON July 6, 2022, 9:26 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis to assess response to immunotherapy for malignant brain PR:PROJECT_TITLE tumors: Part 3 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 Applied Physiology and Kinesiology PR:LABORATORY Rm 042 PR:LAST_NAME Khattri PR:FIRST_NAME Ram PR:ADDRESS 1864 Stadium RD, Gainesville FL 32611 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 3 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 Applied Physiology and Kinesiology ST:LABORATORY Rm 042 ST:LAST_NAME Khattri ST:FIRST_NAME Ram ST:ADDRESS 1200 Newell Dr., ARB 240, Gainesville, FL, 32611, USA ST:EMAIL rbk11@ufl.edu ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 24 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 3307856045 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:GENDER Not applicable 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_serum_Control-1 Group:Control RAW_FILE_NAME=Maryam_serum_Control-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_Control-2 Group:Control RAW_FILE_NAME=Maryam_serum_Control-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_Control-3 Group:Control RAW_FILE_NAME=Maryam_serum_Control-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_Control-4 Group:Control RAW_FILE_NAME=Maryam_serum_Control-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_Control-5 Group:Control RAW_FILE_NAME=Maryam_serum_Control-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_24hrs-1 Group:PostV_24hrs RAW_FILE_NAME=Maryam_serum_PostV_24hrs-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_24hrs-2 Group:PostV_24hrs RAW_FILE_NAME=Maryam_serum_PostV_24hrs-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_24hrs-3 Group:PostV_24hrs RAW_FILE_NAME=Maryam_serum_PostV_24hrs-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_24hrs-4 Group:PostV_24hrs RAW_FILE_NAME=Maryam_serum_PostV_24hrs-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_24hrs-5 Group:PostV_24hrs RAW_FILE_NAME=Maryam_serum_PostV_24hrs-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_48hrs-1 Group:PostV_48hrs RAW_FILE_NAME=Maryam_serum_PostV_48hrs-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_48hrs-2 Group:PostV_48hrs RAW_FILE_NAME=Maryam_serum_PostV_48hrs-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_48hrs-3 Group:PostV_48hrs RAW_FILE_NAME=Maryam_serum_PostV_48hrs-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_48hrs-4 Group:PostV_48hrs RAW_FILE_NAME=Maryam_serum_PostV_48hrs-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_48hrs-5 Group:PostV_48hrs RAW_FILE_NAME=Maryam_serum_PostV_48hrs-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_7days-1 Group:PostV_7 days RAW_FILE_NAME=Maryam_serum_PostV_7days-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_7days-2 Group:PostV_7 days RAW_FILE_NAME=Maryam_serum_PostV_7days-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_7days-3 Group:PostV_7 days RAW_FILE_NAME=Maryam_serum_PostV_7days-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_7days-4 Group:PostV_7 days RAW_FILE_NAME=Maryam_serum_PostV_7days-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_7days-5 Group:PostV_7 days RAW_FILE_NAME=Maryam_serum_PostV_7days-5.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_Day0-1 Group:PostV_Day0 RAW_FILE_NAME=Maryam_serum_PostV_Day0-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_Day0-2 Group:PostV_Day0 RAW_FILE_NAME=Maryam_serum_PostV_Day0-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_Day0-3 Group:PostV_Day0 RAW_FILE_NAME=Maryam_serum_PostV_Day0-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PostV_Day0-4 Group:PostV_Day0 RAW_FILE_NAME=Maryam_serum_PostV_Day0-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PreV_Day0-1 Group:PreV_Day0 RAW_FILE_NAME=Maryam_serum_PreV_Day0-1.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PreV_Day0-2 Group:PreV_Day0 RAW_FILE_NAME=Maryam_serum_PreV_Day0-2.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PreV_Day0-3 Group:PreV_Day0 RAW_FILE_NAME=Maryam_serum_PreV_Day0-3.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PreV_Day0-4 Group:PreV_Day0 RAW_FILE_NAME=Maryam_serum_PreV_Day0-4.raw SUBJECT_SAMPLE_FACTORS - Maryam_serum_PreV_Day0-5 Group:PreV_Day0 RAW_FILE_NAME=Maryam_serum_PreV_Day0-5.raw #COLLECTION CO:COLLECTION_SUMMARY Each animal was in a separate chamber individually. The chamber bottom was CO:COLLECTION_SUMMARY covered with clean parafilm. Animals were kept in the chamber until urination. CO:COLLECTION_SUMMARY Urine drops were collected using sterile syringes. Samples were stored at -80 CO:COLLECTION_SUMMARY °C until further analysis. Serum samples were collected from peripheral blood. CO:COLLECTION_SUMMARY Blood was drawn from the facial vein and was kept in the room temperature for 30 CO:COLLECTION_SUMMARY minutes to coagulate. Coagulated blood was centrifuged at 1500 rpm for 15 CO:COLLECTION_SUMMARY minutes at 4°C. Then serum was collected from the top and was stored at -20 °C CO:COLLECTION_SUMMARY until further analysis. CO:SAMPLE_TYPE Blood (serum) CO:COLLECTION_METHOD Each animal was in a separate chamber individually. The chamber bottom was CO:COLLECTION_METHOD covered with clean parafilm. Animals were kept in the chamber until urination. CO:COLLECTION_METHOD Urine drops were collected using sterile syringes. Samples were stored at -80 CO:COLLECTION_METHOD °C until further analysis. Serum samples were collected from peripheral blood. CO:COLLECTION_METHOD Blood was drawn from the facial vein and was kept in the room temperature for 30 CO:COLLECTION_METHOD minutes to coagulate. Coagulated blood was centrifuged at 1500 rpm for 15 CO:COLLECTION_METHOD minutes at 4°C. Then serum was collected from the top and was stored at -20 °C CO:COLLECTION_METHOD until further analysis. CO:COLLECTION_LOCATION University of Florida, Neurosurgery Department, Medical college, University of CO:COLLECTION_LOCATION Florida CO:COLLECTION_FREQUENCY Pre-vaccination, post vaccination day0, 24 hrs, 48 hrs of post DC vaccination CO:COLLECTION_FREQUENCY and after 7 days. CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS cryovials CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY "DC vaccine treatment Naïve C57/BL6 mice were used for tumor implantation. TR:TREATMENT_SUMMARY B16F10-OVA (2 × 104 cells/brain) tumor cell lines were injected intracranially. TR:TREATMENT_SUMMARY Antigen specific T cells (3 × 107 cells/50 µL of PBS) were infused into the TR:TREATMENT_SUMMARY animals intravenous (IV) five days after tumor implantation and the DC vaccine TR:TREATMENT_SUMMARY (1x106 cells/50 µL of PBS per animal) was injected intradermally on the same TR:TREATMENT_SUMMARY day. Both antigen-specific T cells and DC vaccine were administered once. Urine TR:TREATMENT_SUMMARY samples were collected after DC vaccination at several timepoints. Finally, TR:TREATMENT_SUMMARY animals were euthanized when they reached the endpoints. Sample collection Each TR:TREATMENT_SUMMARY animal was in a separate chamber individually. The chamber bottom was covered TR:TREATMENT_SUMMARY with clean parafilm. Animals were kept in the chamber until urination. Urine TR:TREATMENT_SUMMARY drops were collected using sterile syringes. Samples were stored at -80 °C TR:TREATMENT_SUMMARY until further analysis. Serum samples were collected from peripheral blood. " 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 To isolate metabolites from serum samples, perchloric acid (PCA) extraction was SP:SAMPLEPREP_SUMMARY performed. Fifty microliters of serum sample was taken in a vial and 1 mL of ice SP:SAMPLEPREP_SUMMARY cold 6 % (v/v) PCA was added to it. The mixture was vortexed for 2 minutes and SP:SAMPLEPREP_SUMMARY centrifuged at 4 oC and 13.2 K rpm speed. Supernatant from the centrifuged SP:SAMPLEPREP_SUMMARY sample was taken in a new vial and neutralized with 5 M potassium hydroxide SP:SAMPLEPREP_SUMMARY making its pH near to 6.5. Centrifugation (4 oC temperature, 13.2 K rpm speed) SP:SAMPLEPREP_SUMMARY of the neutralized solution was performed to remove the potassium perchlorate SP:SAMPLEPREP_SUMMARY salt. The supernatant was dried using a room temperature lyophilizer SP:SAMPLEPREP_SUMMARY (Thermo-Scentific, Dallas, USA). The dried powder was suspended in 200 µL of SP:SAMPLEPREP_SUMMARY ultra-pure water. The pH of the mixture was adjusted to 7.0 using 1M sodium SP:SAMPLEPREP_SUMMARY hydroxide and 1M hydrochloride. Centrifugation (4 oC temperature, 13.2 K rpm SP:SAMPLEPREP_SUMMARY speed) of the mixture was performed to remove excess insoluble salt. The SP:SAMPLEPREP_SUMMARY supernatant solution was again lyophilized. The resulted dried powder was SP:SAMPLEPREP_SUMMARY resuspended in deuterated phosphate buffer system to prepare the NMR samples. In SP:SAMPLEPREP_SUMMARY case of serum samples, the solid was dissolved in a 90/10 mixture of 50 mM SP:SAMPLEPREP_SUMMARY sodium phosphate buffer (pH 7.0) with 2 mM of ethylene diamine tetra acetic acid SP:SAMPLEPREP_SUMMARY (EDTA) and a Chenomx internal standard (Chenomx Inc., Alberta, Canada) SP:SAMPLEPREP_SUMMARY consisting of 5 mM 2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt (DSS-D6) SP:SAMPLEPREP_SUMMARY and 0.2% NaN3 in D2O. SP:SAMPLEPREP_PROTOCOL_FILENAME 1. DC-Vaccine-treated-NMR serum Procedures SP:PROCESSING_METHOD Lyophilization SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Perchloric acid extraction method SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION In 500 microliter of 150 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS SP:SAMPLE_RESUSPENSION and 0.2% sodium azide for serum samples. SP:SAMPLE_SPIKING 0.5 mM DSS 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 4/18/2017 AN:ACQUISITION_PARAMETERS_FILE 1. DC-Vaccine-treated-NMR serum Procedures AN:PROCESSING_PARAMETERS_FILE 1. DC-Vaccine-treated-NMR serum 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 0.5mM DSS NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 5mm CPTXI 1H/D -13C/15N Z-GRD Z44866/0026 NM:NMR_SOLVENT Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 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 PROJECT NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 512 NM:OFFSET_FREQUENCY 4.78 ppm NM:CHEMICAL_SHIFT_REF_CPD DSS NM:TEMPERATURE 298.2 oK NM:NUMBER_OF_SCANS 64 NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 4.54s NM:RELAXATION_DELAY 3s NM:SPECTRAL_WIDTH 7211.5 Hz NM:NUM_DATA_POINTS_ACQUIRED 32768 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 DSS NM:BINNED_INCREMENT 0.001 ppm NM:BINNED_DATA_NORMALIZATION_METHOD Largest Peak NM:BINNED_DATA_CHEMICAL_SHIFT_RANGE [0.5,9.5] ppm NM:BINNED_DATA_EXCLUDED_RANGE >9.5 ppm and < 0.5 ppm regions NM:NMR_RESULTS_FILE ST002209_AN003612_Results.txt UNITS:Peak intensity #END