#METABOLOMICS WORKBENCH ssah9_20220210_094443 DATATRACK_ID:3078 STUDY_ID:ST002083 ANALYSIS_ID:AN003400 PROJECT_ID:PR001323 VERSION 1 CREATED_ON February 10, 2022, 10:33 am #PROJECT PR:PROJECT_TITLE Time-Resolved Metabolomics of a Mouse Model of High-Grade Serous Ovarian Cancer PR:PROJECT_TYPE UHPLC-MS and mass spec imaging study of ovarian cancer PR:PROJECT_SUMMARY The dismally low survival rate of ovarian cancer patients diagnosed with PR:PROJECT_SUMMARY high-grade serous carcinoma (HGSC) emphasizes the lack of effective screening PR:PROJECT_SUMMARY strategies. One major obstacle is the limited knowledge of the underlying PR:PROJECT_SUMMARY mechanisms of HGSC pathogenesis at very early stages. Here, we present the first PR:PROJECT_SUMMARY 10-month time-resolved serum metabolic profile of a triple mutant (TKO) HGSC PR:PROJECT_SUMMARY mouse model, along with the spatial lipidome profile of its entire reproductive PR:PROJECT_SUMMARY system. A high-coverage liquid chromatography mass spectrometry-based PR:PROJECT_SUMMARY metabolomics approach was applied to longitudinally collected serum samples from PR:PROJECT_SUMMARY both TKO and TKO control mice, tracking metabolome and lipidome changes from PR:PROJECT_SUMMARY disease onset until mouse death. Spatial lipid distributions within the PR:PROJECT_SUMMARY reproductive system were also mapped via ultrahigh-resolution matrix-assisted PR:PROJECT_SUMMARY laser desorption/ionization (MALDI) mass spectrometry and compared with serum PR:PROJECT_SUMMARY lipid profiles for various lipid classes. Altogether, our results show that the PR:PROJECT_SUMMARY remodeling of lipid and fatty acid metabolism, amino acid biosynthesis, TCA PR:PROJECT_SUMMARY cycle and ovarian steroidogenesis are critical components of HGSC onset and PR:PROJECT_SUMMARY development. These metabolic alterations are accompanied by changes in energy PR:PROJECT_SUMMARY metabolism, mitochondrial and peroxisomal function, redox homeostasis, and PR:PROJECT_SUMMARY inflammatory response, collectively supporting tumorigenesis. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT School of Chemistry & Biochemistry PR:LABORATORY Facundo M. Fernandez PR:LAST_NAME Sah PR:FIRST_NAME Samyukta PR:ADDRESS School of Chemistry & Biochemistry, 901 Atlantic Dr PR:EMAIL ssah9@gatech.edu PR:PHONE 5746780124 #STUDY ST:STUDY_TITLE Time-Resolved Metabolomics of a Mouse Model of High-Grade Serous Ovarian Cancer ST:STUDY_TITLE (MSI) ST:STUDY_SUMMARY The dismally low survival rate of ovarian cancer patients diagnosed with ST:STUDY_SUMMARY high-grade serous carcinoma (HGSC) emphasizes the lack of effective screening ST:STUDY_SUMMARY strategies. One major obstacle is the limited knowledge of the underlying ST:STUDY_SUMMARY mechanisms of HGSC pathogenesis at very early stages. Here, we present the first ST:STUDY_SUMMARY 10-month time-resolved serum metabolic profile of a triple mutant (TKO) HGSC ST:STUDY_SUMMARY mouse model, along with the spatial lipidome profile of its entire reproductive ST:STUDY_SUMMARY system. A high-coverage liquid chromatography mass spectrometry-based ST:STUDY_SUMMARY metabolomics approach was applied to longitudinally collected serum samples from ST:STUDY_SUMMARY both TKO and TKO control mice, tracking metabolome and lipidome changes from ST:STUDY_SUMMARY disease onset until mouse death. Spatial lipid distributions within the ST:STUDY_SUMMARY reproductive system were also mapped via ultrahigh-resolution matrix-assisted ST:STUDY_SUMMARY laser desorption/ionization (MALDI) mass spectrometry and compared with serum ST:STUDY_SUMMARY lipid profiles for various lipid classes. Altogether, our results show that the ST:STUDY_SUMMARY remodeling of lipid and fatty acid metabolism, amino acid biosynthesis, TCA ST:STUDY_SUMMARY cycle and ovarian steroidogenesis are critical components of HGSC onset and ST:STUDY_SUMMARY development. These metabolic alterations are accompanied by changes in energy ST:STUDY_SUMMARY metabolism, mitochondrial and peroxisomal function, redox homeostasis, and ST:STUDY_SUMMARY inflammatory response, collectively supporting tumorigenesis. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT School of Chemistry & Biochemistry ST:LABORATORY Facundo M. Fernandez ST:LAST_NAME Sah ST:FIRST_NAME Samyukta ST:ADDRESS School of Chemistry & Biochemistry, 901 Atlantic Dr ST:EMAIL ssah9@gatech.edu ST:PHONE 5746780124 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #FACTORS #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 - T6_10 Phenotype:TKO RAW_FILE_NAME=TKO 14-7529-6; Subject=Mouse; Collection Date (MM/DD/YYYY)=10/18/2019 SUBJECT_SAMPLE_FACTORS - C1_17 Phenotype:Control RAW_FILE_NAME=Control 7244-12; Subject=Mouse; Collection Date (MM/DD/YYYY)=12/20/2019 #COLLECTION CO:COLLECTION_SUMMARY Blood samples were collected from 17 TKO mice and 16 TKO control mice starting CO:COLLECTION_SUMMARY at 8 weeks of age. A sequential blood sampling procedure was conducted and CO:COLLECTION_SUMMARY samples from each mouse were collected every two weeks until end point or CO:COLLECTION_SUMMARY ascites. Two TKO mice died after 14 and 16 weeks, and were not included in the CO:COLLECTION_SUMMARY UHPLC-MS analysis. One TKO control mouse died after 10 weeks and was also CO:COLLECTION_SUMMARY excluded. For imaging experiments, TKO mice were sacrificed at advanced cancer CO:COLLECTION_SUMMARY stages, their reproductive systems collected and stored at -80 oC for tissue CO:COLLECTION_SUMMARY embedding and sectioning. CO:SAMPLE_TYPE Ovary #TREATMENT TR:TREATMENT_SUMMARY p53LSL R172H/+ Dicer1flox/flox Ptenflox/flox Amhr2cre/+ mice were generated by TR:TREATMENT_SUMMARY mating p53LSL-R172H/+Dicer1flox/floxPtenflox/flox female mice with TR:TREATMENT_SUMMARY Dicer1flox/floxPtenflox/floxAmhr2cre/+ male mice. TR:TREATMENT_SUMMARY p53LSL-R172H/+Dicer1flox/floxPtenflox/flox mice were used as TKO controls TR:TREATMENT_SUMMARY (ctrl). TKO ctrl mice carry the same genetic background as TKO mice but do not TR:TREATMENT_SUMMARY develop HGSC. p53LSL R172H/+ Dicer1flox/flox Ptenflox/flox Amhr2cre/+ mice were TR:TREATMENT_SUMMARY sacrificed in accordance to the animal protocol approved by the institutional TR:TREATMENT_SUMMARY animal care and use committee (IACUC) at Indiana University. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For MS imaging experiments, TKO mice were sacrificed at advanced cancer stages, SP:SAMPLEPREP_SUMMARY their reproductive systems collected and stored at -80 oC. Following examination SP:SAMPLEPREP_SUMMARY of a variety of tissue samples, we focused on a TKO mouse reproductive system SP:SAMPLEPREP_SUMMARY that showed a HGSC on one of the fallopian tubes, with the healthy ovary SP:SAMPLEPREP_SUMMARY engulfed in the tumor and adjacent cysts. In this tissue sample, the HGSC region SP:SAMPLEPREP_SUMMARY connects with the opposite healthy ovary and fallopian tube through the uterus. SP:SAMPLEPREP_SUMMARY These freshly frozen tissues were embedded in an aqueous solution containing 1 % SP:SAMPLEPREP_SUMMARY CMC and 5 % (by weight) gelatin. Tissue embedding was conducted in an SP:SAMPLEPREP_SUMMARY isopentane-dry ice bath at -20 oC. A CryoStar NX70 Cryostat was used for SP:SAMPLEPREP_SUMMARY cryosectioning. The sectioning temperature was set to -20 oC and each slice was SP:SAMPLEPREP_SUMMARY sectioned at a thickness of 10 µm. Sectioned tissue slices were transferred to SP:SAMPLEPREP_SUMMARY Fisherbrand™ Superfrost™ Plus microscope slides for MALDI imaging MS SP:SAMPLEPREP_SUMMARY experiments. Mounted tissue slices were sprayed with 5 mg mL-1 1,5-DAN prior to SP:SAMPLEPREP_SUMMARY MALDI MS. 1,5-DAN was dissolved in 65/20/15 (v/v/v) SP:SAMPLEPREP_SUMMARY acetonitrile/methanol/chloroform and sprayed via an iMatrix matrix sprayer. The SP:SAMPLEPREP_SUMMARY sprayer nozzle height was set to 60 mm, the speed of the nozzle movement was 200 SP:SAMPLEPREP_SUMMARY mm s-1. The inter-line distance was 1 mm. One µL of the matrix solution was SP:SAMPLEPREP_SUMMARY sprayed onto an area of 1 cm2 on average. The spray cycle was repeated 10 times SP:SAMPLEPREP_SUMMARY to ensure complete and uniform matrix coverage on tissue sections. A Bruker SP:SAMPLEPREP_SUMMARY SolariX 12-Tesla Fourier-transform ion cyclotron resonance (FTICR) mass SP:SAMPLEPREP_SUMMARY spectrometer equipped with a MALDI ion source was used for all spatial SP:SAMPLEPREP_SUMMARY lipidomics imaging experiments. The mass spectrometer was operated in the SP:SAMPLEPREP_SUMMARY negative ion mode for fatty acid and lipid feature discovery in the 150–1200 SP:SAMPLEPREP_SUMMARY m/z range. The laser power was set to 25%, and the number of laser shots SP:SAMPLEPREP_SUMMARY accumulated on each pixel was 300. The laser repetition frequency was 1000 Hz, SP:SAMPLEPREP_SUMMARY and the laser beam focus size was set to minimum. The spatial resolution defined SP:SAMPLEPREP_SUMMARY by the pixel size of the images was 50 µm × 50 µm. The time domain data set SP:SAMPLEPREP_SUMMARY size was set to 4,000,000, corresponding to a mass resolution of 410,000 at m/z SP:SAMPLEPREP_SUMMARY 400, and the FID transient time was 0.4194 s. The mass spectrometer was SP:SAMPLEPREP_SUMMARY calibrated externally with (+)ESI and (-)ESI CalMix solution and internally with SP:SAMPLEPREP_SUMMARY FA(18:1) and PI(38:4) to ensure mass accuracy was better than 1 ppm on average. SP:SAMPLEPREP_SUMMARY Observed ions in the average mass MALDI spectrum were subject to Lipid Maps and SP:SAMPLEPREP_SUMMARY HMDB database searches using METASPACE. Features with a false discovery rate of SP:SAMPLEPREP_SUMMARY 10% or less were chosen and compared to features annotated in LC-MS serum SP:SAMPLEPREP_SUMMARY studies. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Solarix FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR MS:MS_TYPE MALDI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data processed by SCiLS Lab Core 2021c, features assigned by METASPACE database MS:MS_RESULTS_FILE ST002083_AN003400_Results.txt UNITS:mass peak abundances Has m/z:Yes Has RT:No RT units:No RT data #END