#METABOLOMICS WORKBENCH Givenchy_20250326_085227 DATATRACK_ID:5787 STUDY_ID:ST003857 ANALYSIS_ID:AN006338 PROJECT_ID:PR002414
VERSION             	1
CREATED_ON             	April 11, 2025, 11:43 am
#PROJECT
PR:PROJECT_TITLE                 	Spatial lipidomics with in situ segmentation for profiling the effect of the
PR:PROJECT_TITLE                 	drug doxorubicin on hepatoma carcinoma cell spheroids
PR:PROJECT_TYPE                  	Mass spectometry imaging
PR:PROJECT_SUMMARY               	This study developed a simple method to analyze lipid changes in 3D liver cancer
PR:PROJECT_SUMMARY               	cell spheroids after treatment with the drug doxorubicin (DOX). Using
PR:PROJECT_SUMMARY               	high-resolution MALDI mass spectrometry imaging (MALDI-MSI) and tissue analysis,
PR:PROJECT_SUMMARY               	the researchers mapped spatial lipid distribution across different regions of
PR:PROJECT_SUMMARY               	the spheroids: the outer proliferative zone, intermediate quiescent zone, and
PR:PROJECT_SUMMARY               	inner necrotic core. They identified distinct lipid alterations in each region
PR:PROJECT_SUMMARY               	(71, 60, and 56 lipids, respectively), with glycerophospholipid metabolism being
PR:PROJECT_SUMMARY               	the most affected pathway. These changes, particularly in PA, PC, PI, and SM
PR:PROJECT_SUMMARY               	lipids, may inhibit necrotic core formation. This spatial lipidomics approach
PR:PROJECT_SUMMARY               	provides insights into the effects of DOX and could help optimize personalized
PR:PROJECT_SUMMARY               	cancer therapies using tumor models.
PR:INSTITUTE                     	Shanxi Medical University
PR:DEPARTMENT                    	School of Basic Medical Sciences, Academy of Medical Sciences, Research
PR:DEPARTMENT                    	Institute of Circadian Rhythm and Disease, Shanxi Medical University
PR:LABORATORY                    	Shanxi Provincial Key Laboratory of Nanoimaging and Drug-Loaded Preparations
PR:LAST_NAME                     	Zhao
PR:FIRST_NAME                    	Huifang
PR:ADDRESS                       	56 Xinjian South Road, Yingze District, Taiyuan, Shanxi Province, China,
PR:ADDRESS                       	Taiyuan, Shanxi Province, 030012, China
PR:EMAIL                         	zhaohf1108@sxmu.edu.cn
PR:PHONE                         	0351-4135004
PR:FUNDING_SOURCE                	National Natural Science Foundation of China
#STUDY
ST:STUDY_TITLE                   	Spatial lipidomics with in situ segmentation for profiling the effect of the
ST:STUDY_TITLE                   	drug doxorubicin on hepatoma carcinoma cell spheroids
ST:STUDY_TYPE                    	Mammal
ST:STUDY_SUMMARY                 	This study developed a simple method to analyze lipid changes in 3D liver cancer
ST:STUDY_SUMMARY                 	cell spheroids after treatment with the drug doxorubicin (DOX). The changes of
ST:STUDY_SUMMARY                 	spatial lipid molecules in 3D Hep-G2 cell spheroids model before and after DOX
ST:STUDY_SUMMARY                 	treatment were analyzed by MALDI-MSI. Using high-resolution MALDI mass
ST:STUDY_SUMMARY                 	spectrometry imaging (MALDI-MSI) and tissue analysis, the researchers mapped
ST:STUDY_SUMMARY                 	spatial lipid distribution across different regions of the spheroids: the outer
ST:STUDY_SUMMARY                 	proliferative zone, intermediate quiescent zone, and inner necrotic core. They
ST:STUDY_SUMMARY                 	identified distinct lipid alterations in each region (71, 60, and 56 lipids,
ST:STUDY_SUMMARY                 	respectively), with glycerophospholipid metabolism being the most affected
ST:STUDY_SUMMARY                 	pathway. These changes, particularly in PA, PC, PI, and SM lipids, may inhibit
ST:STUDY_SUMMARY                 	necrotic core formation. This spatial lipidomics approach provides insights into
ST:STUDY_SUMMARY                 	the effects of DOX and could help optimize personalized cancer therapies using
ST:STUDY_SUMMARY                 	tumor models.
ST:INSTITUTE                     	Shanxi Medical University
ST:DEPARTMENT                    	School of Basic Medical Sciences, Academy of Medical Sciences, Research
ST:DEPARTMENT                    	Institute of Circadian Rhythm and Disease
ST:LABORATORY                    	Shanxi Provincial Key Laboratory of Nanoimaging and Drug-Loaded Preparations
ST:LAST_NAME                     	Zhao
ST:FIRST_NAME                    	Huifang
ST:ADDRESS                       	56 Xinjian South Road, Yingze District, Taiyuan, Shanxi Province, China
ST:EMAIL                         	zhaohf1108@sxmu.edu.cn
ST:PHONE                         	0351-4135004
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	Human HepG2 cells	20240407-DOX-1-Ctrl-1-POS-10um	Sample source:HepG2 cancer cells | Treatment:control	RAW_FILE_NAME(Raw data file names)=20240407-DOX-1-Ctrl-1-POS-10um.d
SUBJECT_SAMPLE_FACTORS           	Human HepG2 cells	20240407-DOX-1-Ctrl-2-POS-10um	Sample source:HepG2 cancer cells | Treatment:control	RAW_FILE_NAME(Raw data file names)=20240407-DOX-1-Ctrl-2-POS-10um.d
SUBJECT_SAMPLE_FACTORS           	Human HepG2 cells	20240407-DOX-1-Ctrl-3-POS-10um	Sample source:HepG2 cancer cells | Treatment:control	RAW_FILE_NAME(Raw data file names)=20240407-DOX-1-Ctrl-3-POS-10um.d
SUBJECT_SAMPLE_FACTORS           	Human HepG2 cells	20240407-DOX-1-POS-10um	Sample source:HepG2 cancer cells | Treatment:DOX treatment	RAW_FILE_NAME(Raw data file names)=20240407-DOX-1-POS-10um.d
SUBJECT_SAMPLE_FACTORS           	Human HepG2 cells	20240407-DOX-2-POS-10um	Sample source:HepG2 cancer cells | Treatment:DOX treatment	RAW_FILE_NAME(Raw data file names)=20240407-DOX-2-POS-10um.d
SUBJECT_SAMPLE_FACTORS           	Human HepG2 cells	20240407-DOX-3-POS-10um	Sample source:HepG2 cancer cells | Treatment:DOX treatment	RAW_FILE_NAME(Raw data file names)=20240407-DOX-3-POS-10um.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Three-dimensional tumor cell spheroids (3D TCS) were cultured using ultra-low
CO:COLLECTION_SUMMARY            	attachment (ULA) 96-well plates. In brief, each well was seeded with 100 µL of
CO:COLLECTION_SUMMARY            	HepG2 cell suspension (~10,000 cells). After 48 hours of incubation, half of the
CO:COLLECTION_SUMMARY            	culture medium was removed and replenished with fresh medium every two days.
CO:COLLECTION_SUMMARY            	Following 7 days of culture, the 3D TCS were treated with DOX. The
CO:COLLECTION_SUMMARY            	DOX-containing medium was then carefully aspirated, and the spheroids were
CO:COLLECTION_SUMMARY            	rinsed three times with PBS. Finally, the 3D TCS were frozen at -80°C for
CO:COLLECTION_SUMMARY            	further analysis.
CO:SAMPLE_TYPE                   	HepG2 human hepatocellular carcinoma cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Treatment Methods: The 3D tumor cell spheroids (TCS) were exposed to DOX at dose
TR:TREATMENT_SUMMARY             	of 329.5 µM for 24 hours. Once the two-dimensional (2D) cells reached 80%
TR:TREATMENT_SUMMARY             	confluence, they were transferred into the chamber shell from the ITO slide.
TR:TREATMENT_SUMMARY             	Subsequently, the 2D cells were exposed to DOX at dose of 1797 nM for 24 hours.
TR:TREATMENT_COMPOUND            	doxorubicin (DOX)
TR:TREATMENT_DOSEDURATION        	24 hours
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The 3D tumor cell spheroids (TCS) were embedded in carboxymethylcellulose (CMC)
SP:SAMPLEPREP_SUMMARY            	and stored at -20 °C for 40 min. The frozen cell spheroids were sectioned into
SP:SAMPLEPREP_SUMMARY            	10 µm thick slices at -20 °C by LEICA CM 1950 cryostat. The slices were
SP:SAMPLEPREP_SUMMARY            	quickly affixed to the ITO slides. A final concentration of 15 mg/mL DHB
SP:SAMPLEPREP_SUMMARY            	dissolved in ACN, water, and TFA (90:10:0.1, V/V/V) was served as the
SP:SAMPLEPREP_SUMMARY            	positive-ion mode matrix and sprayed by the HTX™-Sprayer™ (HTX Technologies,
SP:SAMPLEPREP_SUMMARY            	Carrboro, NC).
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	None (Direct infusion)
CH:INSTRUMENT_NAME               	none
CH:COLUMN_NAME                   	none
CH:SOLVENT_A                     	none
CH:SOLVENT_B                     	none
CH:FLOW_GRADIENT                 	none
CH:FLOW_RATE                     	none
CH:COLUMN_TEMPERATURE            	none
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Bruker timsTOF fleX
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	MALDI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Fifteen cycles of matrix were sprayed onto indium tin oxide (ITO) slides with a
MS:MS_COMMENTS                   	flow rate of 125 µL/min. The gas pressure used for spraying and the temperature
MS:MS_COMMENTS                   	were individually set to 10 psi and 60 °C. The EPSON Scan software was employed
MS:MS_COMMENTS                   	to scan the slides and obtain the raw images, which were analyzed using timsTOF
MS:MS_COMMENTS                   	fleX (Bruker Daltonics, Bremen, Germany) for MALDI-MSI analysis. The detection
MS:MS_COMMENTS                   	range of m/z was from 500 to 1250, and the spatial resolution of MALDI imaging
MS:MS_COMMENTS                   	was set to 10 μM. SCiLS Lab Version 2023b pro was applied to analyze the raw
MS:MS_COMMENTS                   	imaging data. The differential lipids between control and DOX treatment groups
MS:MS_COMMENTS                   	were screened based on the area under curve (AUC) value ≥ 0.75 and p value <
MS:MS_COMMENTS                   	0.05. The structure of differential lipids was further identified.
MS:MS_COMMENTS                   	Identification of lipid molecules was based on the database within Bruker's
MS:MS_COMMENTS                   	MetaboScape 2024b software.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak intensity
MS_METABOLITE_DATA_START
Samples	20240407-DOX-1-Ctrl-1-POS-10um	20240407-DOX-1-Ctrl-2-POS-10um	20240407-DOX-1-Ctrl-3-POS-10um	20240407-DOX-1-POS-10um	20240407-DOX-2-POS-10um	20240407-DOX-3-POS-10um
Factors	Sample source:HepG2 cancer cells | Treatment:control	Sample source:HepG2 cancer cells | Treatment:control	Sample source:HepG2 cancer cells | Treatment:control	Sample source:HepG2 cancer cells | Treatment:DOX treatment	Sample source:HepG2 cancer cells | Treatment:DOX treatment	Sample source:HepG2 cancer cells | Treatment:DOX treatment
4S,5S-antillatoxin A	586	661	772	0	0	0
LysoPC(18:1)	0	0	0	0	0	0
Raffinose	0	0	0	0	0	0
4'-Methyl-(-)-epigallocatechin 7-glucuronide	342	0	316	0	0	0
Arg Tyr Gln Lys	0	0	346	0	0	0
POV-PC	836	780	969	0	0	0
Malyngamide J	0	315	385	0	0	0
Ser Lys Phe Leu Lys	417	662	789	0	0	344
PAz-PC	302	0	0	0	316	0
NCGC00384912-01_C38H61NO9_(3beta,9xi,16alpha)-3-[(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)oxy]-16-hydroxyolean-12-en-28-oic acid	0	0	0	780	1003	961
phosphatidylethanolamine (15:0/16:0)	0	0	0	0	306	0
PA(16:0/18:2)	0	0	0	469	765	528
PA(16:0/18:1)	0	0	0	990	459	304
SM(d18:0/16:1)	0	0	0	0	0	0
PC(16:0/14:0)	1431	825	1220	0	0	0
NCGC00380734-01_C39H65NO10_2-{6-[5-Hydroxy-3-(2-hydroxy-2-propanyl)-6a,9b-dimethyldodecahydrocyclopenta[f]chromen-7-yl]-4a-methyl-3,4,4a,7,8,8a-hexahydro-2H-chromen-2-yl}-2-propanyl 2-acetamido-2-deoxy-6-O-methylhexopyranoside	0	507	667	0	0	0
PA(18:1/18:1)	427	0	0	0	0	0
SM(d18:1/16:0)	0	1018	0	324	0	0
PC(16:1/16:1)	5420	2535	3821	0	0	0
Ubiquinol 8	816	793	978	428	0	0
PC(14:0/18:1)	0	0	0	347	0	0
PE (15:0-18:1-d7)_791638	0	0	314	0	0	0
PC(18:0/14:0)	0	0	0	750	1140	1530
PC(20:2/14:0)	0	0	0	0	612	772
PC(18:1/16:0)	3683	1450	2312	0	0	495
PC(18:2/16:0)	8382	8082	9636	856	0	922
PC(18:3/18:0)	0	0	0	2438	3079	5857
PC(20:2/16:0)	1571	1095	1389	305	0	0
PC(20:0/16:1)	0	0	0	0	0	0
PC(20:3/16:1)	2316	1610	1979	0	0	0
SM(d18:1/22:0)	0	0	0	0	0	0
PC(22:2/16:1)	462	302	441	0	0	0
SM(d18:1/24:1)	0	0	0	0	0	0
PC(18:1/20:3)	0	0	0	1373	1607	3857
SM(d18:0/24:1)	390	0	0	5373	8978	8366
SM(d18:0/24:0)	0	0	0	0	0	0
TG(18:1/14:0/18:1)	0	0	0	761	1283	1647
TG(18:0/14:0/18:1)	369	0	328	0	0	0
TG(16:0/18:0/18:2)	0	0	0	728	1520	2006
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z
4S,5S-antillatoxin A	504.33865
LysoPC(18:1)	522.34911
Raffinose	527.15183
4'-Methyl-(-)-epigallocatechin 7-glucuronide	535.09092
Arg Tyr Gln Lys	594.33254
POV-PC	594.36919
Malyngamide J	608.38422
Ser Lys Phe Leu Lys	622.40038
PAz-PC	666.42622
NCGC00384912-01_C38H61NO9_(3beta,9xi,16alpha)-3-[(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)oxy]-16-hydroxyolean-12-en-28-oic acid	676.44671
phosphatidylethanolamine (15:0/16:0)	678.49856
PA(16:0/18:2)	695.45388
PA(16:0/18:1)	697.46807
SM(d18:0/16:1)	703.5663
PC(16:0/14:0)	706.52931
NCGC00380734-01_C39H65NO10_2-{6-[5-Hydroxy-3-(2-hydroxy-2-propanyl)-6a,9b-dimethyldodecahydrocyclopenta[f]chromen-7-yl]-4a-methyl-3,4,4a,7,8,8a-hexahydro-2H-chromen-2-yl}-2-propanyl 2-acetamido-2-deoxy-6-O-methylhexopyranoside	708.47236
PA(18:1/18:1)	723.48437
SM(d18:1/16:0)	726.55215
PC(16:1/16:1)	730.52578
Ubiquinol 8	731.59753
PC(14:0/18:1)	732.54496
PE (15:0-18:1-d7)_791638	733.54851
PC(18:0/14:0)	734.55807
PC(20:2/14:0)	758.56026
PC(18:1/16:0)	760.5755
PC(18:2/16:0)	780.54204
PC(18:3/18:0)	784.57072
PC(20:2/16:0)	786.59141
PC(20:0/16:1)	788.60445
PC(20:3/16:1)	804.54084
SM(d18:1/22:0)	809.64062
PC(22:2/16:1)	812.60192
SM(d18:1/24:1)	813.67456
PC(18:1/20:3)	832.57204
SM(d18:0/24:1)	837.67079
SM(d18:0/24:0)	839.68484
TG(18:1/14:0/18:1)	853.71452
TG(18:0/14:0/18:1)	855.72909
TG(16:0/18:0/18:2)	881.74574
METABOLITES_END
#END