#METABOLOMICS WORKBENCH LinShuhai_20250318_035241 DATATRACK_ID:5735 STUDY_ID:ST003878 ANALYSIS_ID:AN006370 PROJECT_ID:PR002432 VERSION 1 CREATED_ON April 27, 2025, 2:28 am #PROJECT PR:PROJECT_TITLE Integrative spatially-resolved multi-omics analysis reveals metabolic evolution PR:PROJECT_TITLE of the transition to invasive breast cancer PR:PROJECT_SUMMARY Tumor metastasis is one of the primary causes of death among cancer patients, PR:PROJECT_SUMMARY including those with breast cancer. We conducted a spatially resolved PR:PROJECT_SUMMARY transcriptomics study to analyze the progression of breast cancer from carcinoma PR:PROJECT_SUMMARY in situ to invasive carcinoma, aiming to identify changes during this evolution. PR:PROJECT_SUMMARY Our analysis revealed that PSAP plays an important role in this process. We PR:PROJECT_SUMMARY performed PSAP knockdown in 4T1 cells to investigate the resulting metabolic PR:PROJECT_SUMMARY changes. The results confirmed that silencing PSAP can influence the growth and PR:PROJECT_SUMMARY metastasis of 4T1 cells through sphingolipid metabolism. PR:INSTITUTE Xiamen university PR:LAST_NAME Shu-hai PR:FIRST_NAME Lin PR:ADDRESS No. 4221 Xiang'an South Road, Xiang'an District, Xiamen City, Fujian Province, PR:ADDRESS Xiamen, Fujian, 361100, China PR:EMAIL shuhai@xmu.edu.cn PR:PHONE 15021534583 #STUDY ST:STUDY_TITLE Integrative spatially-resolved multi-omics analysis reveals metabolic evolution ST:STUDY_TITLE of the transition to invasive breast cancer ST:STUDY_SUMMARY We used MSTI (Mass Spectrometry imaging and spatially-resolved Transcriptomics ST:STUDY_SUMMARY Integrator) framework identified the intricate interplay between sphingolipid ST:STUDY_SUMMARY metabolism and prosaposin (PSAP), highlighting the transition from ductal ST:STUDY_SUMMARY carcinoma in situ (DCIS) to invasive breast cancer (IBC) within the tumor ST:STUDY_SUMMARY microenvironment. To investigate the impact of PSAP and sphingolipid metabolism ST:STUDY_SUMMARY on DCIS evolution, we performed PSAP knockdown in 4T1 cells and conducted LC-MS ST:STUDY_SUMMARY analysis to compare the metabolic profiles of wild-type (WT) and PSAP-knockdown ST:STUDY_SUMMARY cells. LC-MS results revealed significant metabolic alterations: compared to ST:STUDY_SUMMARY 4T1-shCtrl cells, 4T1-shPSAP cells exhibited a marked increase in various ST:STUDY_SUMMARY ceramides, a significant upregulation of hexosylceramides (HexCer), and a ST:STUDY_SUMMARY notable decrease in other sphingolipids. These findings highlight the key role ST:STUDY_SUMMARY of PSAP in modulating sphingolipid metabolism during tumor progression. ST:INSTITUTE Xiamen university ST:LAST_NAME Shu-hai ST:FIRST_NAME Lin ST:ADDRESS No. 4221 Xiang'an South Road, Xiang'an District, Xiamen City, Fujian Province, ST:ADDRESS Xiamen, Fujian, 361100, China ST:EMAIL shuhai@xmu.edu.cn ST:PHONE 15021534583 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - blank_1_pos Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_1_pos.raw SUBJECT_SAMPLE_FACTORS - blank_2_pos Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_2_pos.raw SUBJECT_SAMPLE_FACTORS - blank_3_pos Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_3_pos.raw SUBJECT_SAMPLE_FACTORS - blank_4_pos Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_4_pos.raw SUBJECT_SAMPLE_FACTORS - QC_1_pos Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_1_pos.raw SUBJECT_SAMPLE_FACTORS - QC_2_pos Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_2_pos.raw SUBJECT_SAMPLE_FACTORS - QC_3_pos Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_3_pos.raw SUBJECT_SAMPLE_FACTORS - shCtrl_1_pos Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_1_pos.raw SUBJECT_SAMPLE_FACTORS - shCtrl_2_pos Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_2_pos.raw SUBJECT_SAMPLE_FACTORS - shCtrl_3_pos Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_3_pos.raw SUBJECT_SAMPLE_FACTORS - shCtrl_4_pos Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_4_pos.raw SUBJECT_SAMPLE_FACTORS - shPSAP_1_pos Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_1_pos.raw SUBJECT_SAMPLE_FACTORS - shPSAP_2_pos Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_2_pos.raw SUBJECT_SAMPLE_FACTORS - shPSAP_3_pos Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_3_pos.raw SUBJECT_SAMPLE_FACTORS - shPSAP_4_pos Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_4_pos.raw SUBJECT_SAMPLE_FACTORS - blank_1_neg Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_1_neg.raw SUBJECT_SAMPLE_FACTORS - blank_2_neg Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_2_neg.raw SUBJECT_SAMPLE_FACTORS - blank_3_neg Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_3_neg.raw SUBJECT_SAMPLE_FACTORS - blank_4_neg Sample source:blank | Variant:blank sample RAW_FILE_NAME(Raw file name)=blank_4_neg.raw SUBJECT_SAMPLE_FACTORS - QC_1_neg Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_1_neg.raw SUBJECT_SAMPLE_FACTORS - QC_2_neg Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_2_neg.raw SUBJECT_SAMPLE_FACTORS - QC_3_neg Sample source:breast cancer cells | Variant:QC RAW_FILE_NAME(Raw file name)=QC_3_neg.raw SUBJECT_SAMPLE_FACTORS - shCtrl_1_neg Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_1_neg.raw SUBJECT_SAMPLE_FACTORS - shCtrl_2_neg Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_2_neg.raw SUBJECT_SAMPLE_FACTORS - shCtrl_3_neg Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_3_neg.raw SUBJECT_SAMPLE_FACTORS - shCtrl_4_neg Sample source:breast cancer cells | Variant:4T1-shCtrl RAW_FILE_NAME(Raw file name)=shCtrl_4_neg.raw SUBJECT_SAMPLE_FACTORS - shPSAP_1_neg Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_1_neg.raw SUBJECT_SAMPLE_FACTORS - shPSAP_2_neg Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_2_neg.raw SUBJECT_SAMPLE_FACTORS - shPSAP_3_neg Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_3_neg.raw SUBJECT_SAMPLE_FACTORS - shPSAP_4_neg Sample source:breast cancer cells | Variant:4T1-shPSAP RAW_FILE_NAME(Raw file name)=shPSAP_4_neg.raw #COLLECTION CO:COLLECTION_SUMMARY 293T cells and 4T1 breast cancer cells were cultured in dulbecco's modified CO:COLLECTION_SUMMARY eagle medium (DMEM respectively, BasalMedia L120KJ) supplemented with 10% fetal CO:COLLECTION_SUMMARY bovine serum (FBS, VivaCell C04001-500) and 1% pen–strep antibiotic (Beyotime CO:COLLECTION_SUMMARY C0224-100ml). These cells were cultured at 37℃ in a humidified atmosphere CO:COLLECTION_SUMMARY containing 5% CO2. CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY Plasmid/shRNA Construction and Virus Infection: Knockdown cell lines were TR:TREATMENT_SUMMARY generated by lentiviral infection. Briefly, 293T cells were co-transfected with TR:TREATMENT_SUMMARY these plasmids: psPAX2, pMD2.G and pLKO.1 (Hedgehog Bio Science and Technology TR:TREATMENT_SUMMARY Ltd. HH-shRNA-066) containing a shRNA sequence targeting PSAP mRNA. The TR:TREATMENT_SUMMARY following sequences were used: shPSAP 1: 5'- GAACTACGTGGACCAGTATTC-3'; shPSAP 2: TR:TREATMENT_SUMMARY 5'- GAATACTGGTCCACGTAGTTC-3'. The plasmids and shRNA were mixed in FBS-free DMEM TR:TREATMENT_SUMMARY with lipo 3000 kit (Invitrogen L3000015) to form a complex, and incubated with TR:TREATMENT_SUMMARY 293T cell line in FBS-free DMEM in 6-wells plates. The medium was changed with TR:TREATMENT_SUMMARY 10% FBS DMEM after 8h and co-infected in incubator for 48h. After co-infection, TR:TREATMENT_SUMMARY lentiviral supernatants were collected and mixed with virus concentration kit TR:TREATMENT_SUMMARY (Beyotime, C2901L). After 12000g centrifuging for 2min at 4℃, 4T1 cells were TR:TREATMENT_SUMMARY infected with shNC or shPSAP supernatants, as well as 10% FBS DMEM and 10μg/ml TR:TREATMENT_SUMMARY polybrene (Biosharp, BL628A) for 48 h. Stably PSAP knockdown 4T1 cells lines TR:TREATMENT_SUMMARY were selected with 2 μg/ml blasticidin. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipidomic analysis of ceramide-derived lipid species, 1×107 cells were SP:SAMPLEPREP_SUMMARY digested by 0.25% trypsin (Beyotime C0201) and 1000g centrifuged for 3min at SP:SAMPLEPREP_SUMMARY 25℃. Cells were resuspended by 440μl ice-cold 75% methanol (methanol: SP:SAMPLEPREP_SUMMARY dH2O=3:1) with lipid Internal Standard and move the suspension into 2ml EP SP:SAMPLEPREP_SUMMARY tubes. After grinded, the tubes were floated in liquid nitrogen and stewed on SP:SAMPLEPREP_SUMMARY ice for freeze-thawing three times. Then added 1.1ml Methyl tert-Butyl Ether SP:SAMPLEPREP_SUMMARY (MTBE) into the tubes. After quickly shaking the tubes at room temperature, we SP:SAMPLEPREP_SUMMARY added 275μl ddH2O and shook them quickly again. The tubes were centrifuged at SP:SAMPLEPREP_SUMMARY 15000g for 10min at 4℃, then we got the upper layer of the layering into new SP:SAMPLEPREP_SUMMARY 1.5ml EP tubes. The samples were dried under nitrogen, and stored at −80℃ SP:SAMPLEPREP_SUMMARY until LC–MS analysis was performed.All the QCs are pooled samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A: CH2Cl2:MeOH=2:1; Solvent B: 2mM ammonium formate CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Orbitrap Exploris 240 CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) CH:SOLVENT_A 66.67% dichloromethane/33.33% Methanol CH:SOLVENT_B 100% water; 2mM ammonium formate CH:FLOW_GRADIENT The gradient began at 32% B（and 68% A） for 1.5 min, then shifted to 85% B CH:FLOW_GRADIENT for 14min, 97% B for 0.1 min, then stayed at 97% B for 2.4 min before ramping CH:FLOW_GRADIENT down to 32% B for 0.1 min. CH:FLOW_RATE 0.26mL/min CH:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS High-throughput identification and relative quantification of lipids was MS:MS_COMMENTS performed using online product (https://github.com/LinShuhaiLAB/QuanFormer) to MS:MS_COMMENTS search and alignment. The aligned Lipid Ion report was exported to Microsoft MS:MS_COMMENTS Excel. The data whose final quality score, which was calculated by the online MS:MS_COMMENTS product, < 2.1 was excluded. MS:MS_RESULTS_FILE ST003878_AN006370_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END