#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