#METABOLOMICS WORKBENCH TatianaJoao_20230530_001832 DATATRACK_ID:4054 STUDY_ID:ST002720 ANALYSIS_ID:AN004410 PROJECT_ID:PR001307 VERSION 1 CREATED_ON May 30, 2023, 1:24 am #PROJECT PR:PROJECT_TITLE Biochemical Impact of Platinum and Palladium-based Anticancer Agents – PR:PROJECT_TITLE BioIMPACT PR:PROJECT_TYPE NMR-based metabolomics PR:PROJECT_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used PR:PROJECT_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are PR:PROJECT_SUMMARY still current drawbacks. Palladium (Pd(II))-complexes are alternatives due to PR:PROJECT_SUMMARY similar metal coordination and promising cytotoxic properties. Metabolomics can PR:PROJECT_SUMMARY measure the metabolic response of both drug-exposed cells and tissues, unveiling PR:PROJECT_SUMMARY insight into drug mechanisms and metabolic markers of drug efficacy, toxicity PR:PROJECT_SUMMARY and resistance. The present 1H NMR metabolomics study aims to (i) describe the PR:PROJECT_SUMMARY endometabolome of both MDA-MB-231 parental and cDDP-resistant cells PR:PROJECT_SUMMARY (MDA-MB-231R), which are representative of Triple-Negative Breast Cancer, and PR:PROJECT_SUMMARY (ii) characterize the metabolic profile of both cell types exposed to the novel PR:PROJECT_SUMMARY Pd(II)-spermine complex (Pd2Spm), in comparison with cDDP signature, describing PR:PROJECT_SUMMARY possible biomarker patterns of tumor resistance and therapy response. PR:INSTITUTE University of Aveiro PR:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials PR:LABORATORY Metabolomics from Ana M. Gil PR:LAST_NAME Carneiro PR:FIRST_NAME Tatiana J. PR:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal PR:EMAIL tatiana.joao@ua.pt PR:PHONE +351926369478 PR:FUNDING_SOURCE This work was developed within the CICECO-Aveiro Institute of Materials project PR:FUNDING_SOURCE (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) financed by national funds PR:FUNDING_SOURCE through the FCT/MTES (PIDDAC). We also acknowledge funds from POCentro, Portugal PR:FUNDING_SOURCE 2020 and European Community through the FEDER and by the Portuguese Foundation PR:FUNDING_SOURCE for Science and Technology (FCT) through LAQV/REQUIMTE FCT UIDB/50006/2020 PR:FUNDING_SOURCE (C.D.), UIDB/00070/2020 (A.L.M.B.d.C. and M.P.M.M.) and PR:FUNDING_SOURCE POCI-01-0145-FEDER-0016786. We are grateful to the Portuguese National NMR PR:FUNDING_SOURCE Network (PTNMR), supported by FCT funds as the NMR spectrometer used is part of PR:FUNDING_SOURCE PTNMR and partially supported by Infrastructure Project No. 022161 (co-financed PR:FUNDING_SOURCE by FEDER through COMPETE 2020, POCI and PORL, and the FCT through PIDDAC). PR:FUNDING_SOURCE T.J.C. thanks FCT for her Ph.D. grant SFRH/BD/145920/2019 and M.V. thanks the PR:FUNDING_SOURCE FCT and the Ph.D. Program in Med-icines and Pharmaceutical Innovation (i3DU) for PR:FUNDING_SOURCE his Ph.D. grant PD/BD/135460/2017; both grants were funded by the European PR:FUNDING_SOURCE Social Fund of the European Union and national FCT/MCTES funds. #STUDY ST:STUDY_TITLE Metabolic characterization of the polar endometabolome of Triple-Negative Breast ST:STUDY_TITLE Cancer parental and cDDP-resistant cells ST:STUDY_TYPE NMR-based metabolomics ST:STUDY_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used ST:STUDY_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are ST:STUDY_SUMMARY still current drawbacks. Metabolomics can measure the metabolic response of ST:STUDY_SUMMARY drug-exposed cells, unveiling insight into drug mechanisms and metabolic markers ST:STUDY_SUMMARY of drug efficacy, toxicity and resistance. The present 1H NMR metabolomics study ST:STUDY_SUMMARY aims to describe the polar endometabolome of both MDA-MB-231 parental and ST:STUDY_SUMMARY cDDP-resistant cells (MDA-MB-231R), which are representative of Triple-Negative ST:STUDY_SUMMARY Breast Cancer, aiding the current knowledge about the resistant cells metabolism ST:STUDY_SUMMARY rewiring and disclosing metabolic hotspots as possible targets to counteract the ST:STUDY_SUMMARY therapy resistance. ST:INSTITUTE University of Aveiro ST:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials ST:LABORATORY Metabolomics from Ana M. Gil ST:LAST_NAME Carneiro ST:FIRST_NAME Tatiana João ST:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal ST:EMAIL tatiana.joao@ua.pt ST:PHONE +351926369478 ST:NUM_GROUPS 6 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN MDA-MB-231 cells SU:GENDER Not applicable SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC (Manassas, VA, USA); ATCC HTB-26 SU:CELL_STRAIN_DETAILS Epithelial breast cancer cells; absence of estrogen and progesterone receptors, SU:CELL_STRAIN_DETAILS HER2 overexpression SU:CELL_PASSAGE_NUMBER Between 5 to 10 SU:CELL_COUNTS 5 M #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 - R_C0h_EA_11_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_12_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_13_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_21_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_22_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_23_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_31_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_32_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - R_C0h_EA_33_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_11_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_12_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_13_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_21_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_22_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_23_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_31_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_32_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - R_C24h_EA_33_1_2 Treatment_group:Resistant_Controls_24h RAW_FILE_NAME=R_C24h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_11_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_12_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_13_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_21_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_22_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_23b_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_23b_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_31_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_32_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - R_C48h_EA_33_1_2 Treatment_group:Resistant_Controls_48h RAW_FILE_NAME=R_C48h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - S_C0h_EA_11_1_2 Treatment_group:Sensitive_Controls_0h RAW_FILE_NAME=S_C0h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - S_C0h_EA_12_1_2 Treatment_group:Sensitive_Controls_0h RAW_FILE_NAME=S_C0h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - S_C0h_EA_13_1_2 Treatment_group:Sensitive_Controls_0h RAW_FILE_NAME=S_C0h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - 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S_C24h_EA_22_1_2 Treatment_group:Sensitive_Controls_24h RAW_FILE_NAME=S_C24h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_C24h_EA_23_1_2 Treatment_group:Sensitive_Controls_24h RAW_FILE_NAME=S_C24h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_C24h_EA_31_1_2 Treatment_group:Sensitive_Controls_24h RAW_FILE_NAME=S_C24h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_C24h_EA_32_1_2 Treatment_group:Sensitive_Controls_24h RAW_FILE_NAME=S_C24h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_C24h_EA_33_1_2 Treatment_group:Sensitive_Controls_24h RAW_FILE_NAME=S_C24h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_11_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_12_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_13_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_21_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_22_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_23_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_31_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_32_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_33_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_33_1_2 #COLLECTION CO:COLLECTION_SUMMARY MDA-MB-231 parental and resistant (MDA-MB-231/R) cells were seeded at a density CO:COLLECTION_SUMMARY of 3 × 10^4 cells/cm2 onto 13.55 cm diameter Petri dishes, cultured in a CO:COLLECTION_SUMMARY humidified atmosphere of 5% CO2 at 37 ◦C and allowed to adhere for 24 h. After CO:COLLECTION_SUMMARY this, cells were incubated and collected at 0, 24 and 48 h, with basis on the CO:COLLECTION_SUMMARY population (25.5 ± 0.9 h and 30.6 ± 1.1 h for MDA-MB-231 and MDA-MB-231/R CO:COLLECTION_SUMMARY cells, respectively). At each time-point, cells were harvested using a 0.25% CO:COLLECTION_SUMMARY (v/v) trypsin-EDTA solution, washed twice with PBS and centrifuged (300 g, 5 CO:COLLECTION_SUMMARY min, 20 ◦C). The cell pellet was directly stored at − 80 ◦C until CO:COLLECTION_SUMMARY analysis. Three independent experiments with triplicates were performed for each CO:COLLECTION_SUMMARY cell type and time-point. CO:SAMPLE_TYPE Breast cancer cells CO:COLLECTION_DURATION Between 2 and 5 minutes CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY In this study, both cell types (MDA-MB-231 and MDA-MB-231/R) correspond to TR:TREATMENT_SUMMARY controls, so no treatment was applied to these two groups. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cellular polar extracts were extracted using a biphasic extraction method of SP:SAMPLEPREP_SUMMARY methanol/chloroform/water. Basically, cell pellets were resuspended in 650 µL SP:SAMPLEPREP_SUMMARY of 80% (v/v) methanol-miliQ water solution, transferred to microcentrifuge tubes SP:SAMPLEPREP_SUMMARY with 150 mg of glass beads, and vortexed for 5 min. Subsequently, 260 µL of SP:SAMPLEPREP_SUMMARY 100% chloroform and 260 µL of 100% chloroform plus 220 µL MiliQ water were SP:SAMPLEPREP_SUMMARY added to samples, which were vortexed for 5 min between solvents addition. The SP:SAMPLEPREP_SUMMARY samples were kept at − 20 °C for 10 min and centrifuged. The aqueous phase of SP:SAMPLEPREP_SUMMARY the resulting extract was collected into a new tube, vacuum-dried and stored at SP:SAMPLEPREP_SUMMARY − 80 °C until the NMR analysis. Previously to NMR spectra acquision, the dry SP:SAMPLEPREP_SUMMARY aqueous extracts were suspended in 650 µL of 100 mM sodium phosphate buffer (pH SP:SAMPLEPREP_SUMMARY 7.4, in D2O containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) SP:SAMPLEPREP_SUMMARY for chemical shift referencing) and transferred into 5 mm NMR tubes SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Biphasic method (methanol/ chloroform/ water) SP:EXTRACT_STORAGE -80℃ #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics Ana M. Gil AN:SOFTWARE_VERSION Topspin 3.2 AN:ACQUISITION_DATE February 2023 #NMR NM:INSTRUMENT_NAME Avance III TM HD 500MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:NMR_COMMENTS On folder 1 and folder pdata: 1st subfolder contains raw spectra; 2nd subfolder NM:NMR_COMMENTS contains manually processed spectra NM:FIELD_FREQUENCY_LOCK Deuterium NM:SPECTROMETER_FREQUENCY 500MHz NM:NMR_PROBE TXI NM:NMR_SOLVENT D2O NM:NMR_TUBE_SIZE 5mm NM:SHIMMING_METHOD Topshim NM:RECEIVER_GAIN 203 NM:TEMPERATURE 298K NM:NUMBER_OF_SCANS 512 NM:ACQUISITION_TIME 2.34s NM:RELAXATION_DELAY 2s NM:SPECTRAL_WIDTH 7002.801 NM:ZERO_FILLING 64k NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD TSP (3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid) NM:NMR_RESULTS_FILE ST002720_AN004410_Results.txt UNITS:ppm #END