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Imaging was performed using a spatial resolution of 20 um and the minimum laser spot size. A total of 20 laser shots were used per pixel. Data was collected in negative ion mode in the mass range 500-1300. External callibration was performed prior to analysis using the electrospray ion source and NaTFA clusters. FlexImaging (Bruker Daltonics) was used to visualize the imaging data and DataAnalysis was used to visualize individual mass spectra. After identifying cell specific peaks of interest; those peaks were searched out manually in the extreme mass resolution MSI dataset acquired by MALDI FT-ICR (Solarix). The found m/z values were compared with homebuilt lipidomics database provided by Lipometrix (KU Leuven) and putatively identified considering a mass error < 1 ppm.MetaboLightsPublicMS Imaging -The M5 HTX sprayer (HTX technologies; LLC) was used to apply the MALDI matrix N-ethylenediamminonaphtalene hydrochloride (NEDC) (Sigma-Aldrich;N9125-100G) (7g/L in 70% methanol). Matrix was applied using 18 passes; 0.06 mL/min flow rate; 1200 mm/min velocity; 3 mm spacing; 2 L/min N2 flow; CrissCross spray pattern; 40 mm nozzle height and 10 psi. The resulting matrix density was 2.1 mg/mm2. EGF-samples and untreated CTR samples were paired up before matrix application so that each run of matrix application was performed on one EGF-sample and one CTR-sample:Run 1: Samples E13 (EGF) and E18 (CTR)Run 2: Samples E14 (EGF) and E17 (CTR)Run 3: Samples E15 (EGF) and E16 (CTR)Run 4: Samples E22 (EGF) and E21 (CTR)Run 5: Samples E23 (EGF) and E19 (CTR)Run 6: Samples E24 (EGF) and E20 (CTR)Run 7: Samples E28 (EGF) and E27 (CTR)Run 8: Samples E30 (EGF) and E26 (CTR)Run 9: Samples E29 (EGF) and E25 (CTR)Single-cell lipidomic analysis of the Epithelial-Mesenchymal Transition (EMT) using Mass Spectrometry Imaging.Ellen Marie Botne QuinsgaardNorwegian University of Science and TechnologyMDA-MB-468 cellmass spectrometry assayHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS12650Siver Moestue. Department of Clinical and Molecular Medicine, NTNU Trondheim. NTNU Department of Clinical and Molecular Medicine, P.O. Box 8905, 7491 Trondheim, Norway. siver.a.moestue@ntnu.no.Marco Giampa. Metabolomics Expertise Center (VIB-KU Leuven). ON5 Herestraat 49 - box 912, 3000 Leuven, Belgium. marco.giampa@kuleuven.be.Ellen Marie Quinsgaard. Department of Clinical and Molecular Medicine, NTNU Trondheim. embquins@gmail.com.MSI datasets were converted to imzML format in FlexImaging (Bruker Daltonics) and pre-processed using the Cardinal package for R [1,2]. TIC normalization; peak-picking with threshold of signal to noise ratio of 6 and peak-alignment were applied to obtain a centroided processed dataset. Spatial K mean clustering was used to identify cell specific clusters and subset the datasets. [1] Bemis;K.A.;Föll;M.C.;Guo;D. et al. Cardinal v.3: a versatile open-source software for mass spectrometry imaging analysis. Nat Methods20;1883–1886 (2023). https://doi.org/10.1038/s41592-023-02070-z[2] R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing;Vienna;Austria. URL https://www.R-project.org/.Treatmentellen.marie@quinsgaard.comITO-slides (Bruker Daltonik GmbH;8237001) were sterilized in 75% ethanol; rinsed with sterile Phosphate Buffered Saline (PBS) (Sigma-Aldrich;D8537) and placed in 10 cm cell culture dishes. In each dish; 500.000 MDA-MB-468 cells were seeded out in 10 mL DMEM: F12 cell medium with HEPES (Gibco;3133038) supplemented with 5% Fetal Bovine Serum (FBS) (Gibco;10270) and 50 U/mL Penicillin-Streptomycin (P/S) (Gibco;15070063). After 24 h; medium was replaced with FBS-free medium. After another 24 h; cells were stimulated for 72 h with 25 ng/mL Epidermal Growth Factor (EGF) (Peprotech;AF-100-15). After EGF-stimulation; medium was removed and the slides were rinsed with PBS before being fixed with 4% buffered formaldehyde (pH 7; stabilized in 0.5-1.5% methanol) (VWR Chemicals; 9713.1000) for 2 min. The slides were rinsed once with PBS and twice with ammonium acetate (150 mM) (Thermofisher Scientific; R1181) before being dried under a N2-stream. Matrix application was performed within minutes of drying. The M5 HTX sprayer (HTX technologies; LLC) was used to apply the MALDI matrix N-ethylenediamminonaphtalene hydrochloride (NEDC) (Sigma-Aldrich;N9125-100G) (7g/L in 70% methanol). Matrix was applied using 18 passes; 0.06 mL/min flow rate; 1200 mm/min velocity; 3 mm spacing; 2 L/min N2 flow; CrissCross spray pattern; 40 mm nozzle height and 10 psi. The resulting matrix density was 2.1 mg/mm2. EGF-samples and untreated control (CTR) samples were paired up before matrix application so that each run of matrix application was performed on one EGF-sample and one CTR-sample: Run 1: Samples E13 (EGF) and E18 (CTR) Run 2: Samples E14 (EGF) and E17 (CTR) Run 3: Samples E15 (EGF) and E16 (CTR) Run 4: Samples E22 (EGF) and E21 (CTR) Run 5: Samples E23 (EGF) and E19 (CTR) Run 6: Samples E24 (EGF) and E20 (CTR) Run 7: Samples E28 (EGF) and E27 (CTR) Run 8: Samples E30 (EGF) and E26 (CTR) Run 9: Samples E29 (EGF) and E25 (CTR)MetabolomicsNot relevant for this dataset as MALDI MSI was performed on seeded cells rather than tissue sections.After samples were fixed and dried the M5 HTX sprayer (HTX technologies; LLC) was used to apply the MALDI matrix N-ethylenediamminonaphtalene hydrochloride (NEDC) (Sigma-Aldrich;N9125-100G) (7g/L in 70% methanol). Matrix was applied using 18 passes; 0.06 mL/min flow rate; 1200 mm/min velocity; 3 mm spacing; 2 L/min N2 flow; CrissCross spray pattern; 40 mm nozzle height and 10 psi. The resulting matrix density was 2.1 mg/mm2. EGF-samples and untreated CTR samples were paired up before matrix application so that each run of matrix application was performed on one EGF-sample and one CTR-sample:Run 1: Samples E13 (EGF) and E18 (CTR)Run 2: Samples E14 (EGF) and E17 (CTR)Run 3: Samples E15 (EGF) and E16 (CTR)Run 4: Samples E22 (EGF) and E21 (CTR)Run 5: Samples E23 (EGF) and E19 (CTR)Run 6: Samples E24 (EGF) and E20 (CTR)Run 7: Samples E28 (EGF) and E27 (CTR)Run 8: Samples E30 (EGF) and E26 (CTR)Run 9: Samples E29 (EGF) and E25 (CTR)FlexImagingRapiflex MALDI Tissuetyper mass spectrometerMDA-MB-468untargeted analysisBruker Daltonics5.1.46.0_1455_51Homo sapiensnonematrix assisted laser desorption-ionisation mass spectrumexperimental sampleMDA-MB-468 cellepithelial to mesenchymal transitionuntargeted metabolitesepidermal growth factorFlexImagingRapiflex MALDI Tissuetyper mass spectrometerMDA-MB-468untargeted analysisBruker DaltonicsHomo sapiens5.1.46.0_1455_51nonematrix assisted laser desorption-ionisation mass spectrumexperimental sampleMDA-MB-468 cellepithelial to mesenchymal transitionuntargeted metabolitesepidermal growth factorMALDI MSI measurements were performed using a rapifleX MALDI Tissuetyper™ time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). The analyzer was operated in negative reflector mode, and the laser was fired with a repetition rate of 10 kHz using a lateral resolution of 10 µm and 300 shots per pixel. External calibration was performed using red phosphorus dispersed in acetone (100 mg/mL), spotted on a cell-and matrix-free area. Mass spectra were acquired in the m/z range 500–1000 with a digitizer frequency of 1.25 GHz.phosphatidylinositol 36:2(1-)phosphatidylinositol 38:1(1-)phosphatidic acid 34:1phosphatidylglycerol 34:1phosphatidylglycerol 36:1phosphatidylinositol 38:5(1-)phosphatidic acid 36:2phosphatidylethanolamine 38:4 zwitterionphosphatidylinositol 36:3phosphatidylinositol 36:4phosphatidylserine 34:1(1-)phosphatidylinositol 36:1(1-)phosphatidylinositol 34:1(1-)phosphatidylinositol 34:2(1-)phosphatidylserine 36:2phosphatidylglycerol 36:2(1-)phosphatidylinositol 38:4phosphatidylinositol 32:1(1-)phosphatidylethanolamine 36:2 zwitterionphosphatidylethanolamine 34:2 zwitterionphosphatidylinositol 38:3(1-)phosphatidylserine 36:1(1-)LPI(18:0)phosphatidylethanolamine 38:2 zwitterionphosphatidylinositol 32:0(1-)phosphatidylethanolamine 36:1 zwitterionphosphatidylinositol 38:2(1-)phosphatidylethanolamine 36:3falseSingle-cell lipidomic analysis of the Epithelial-Mesenchymal Transition (EMT) using MALDI-MSIEMT is a metastasis-promoting process whose heterogeneity has been extensively studied at a gene expression level. EMT also involves reprogramming of lipid metabolism, however there has been little focus on heterogeneity on the lipid level. Here, we use MALDI-MSI to determine the levels of various glycerophospholipids at the single cell level during EGF-induced EMT in MDA-MB-468 breast cancer cells. Cells undergoing EMT had reduced levels of PA, PS, PE and PI-species, as well as increased levels of PG-species and LPI (18:0). Applying multivariate analysis on the spatially resolved MALDI-data, we found that the metabolic response was heterogeneous, with varying degrees of alteration of lipid-levels after EMT.  Cell organization also  affected lipid regulation, as dispersed cells were more “EMT-like” than cohesive cells.  The fraction of dispersed cells increased substantially during EMT, indicating that pathways which regulate adhesion and motility also regulate lipid metabolism. 2026-04-152025-06-27MTBLS12650MTBLC74465MTBLC134236MTBLC134237MTBLC71721MTBLC141589MTBLC71728MTBLC71727MTBLC136225MTBLC72058MTBLC71737MTBLC71735MTBLC75200MTBLC134490MTBLC136257MTBLC72065MTBLC74369MTBLC74231MTBLC74238MTBLC74237MTBLC136235MTBLC136489MTBLC74372MTBLC74371MTBLC74241MTBLC136239MTBLC91039MTBLC74375MTBLC74374CHEBI:74465CHEBI:134236CHEBI:134237CHEBI:71721CHEBI:141589CHEBI:71728CHEBI:71727CHEBI:136225CHEBI:72058CHEBI:71737CHEBI:71735CHEBI:75200CHEBI:134490CHEBI:136257CHEBI:72065CHEBI:74369CHEBI:74231CHEBI:74238CHEBI:74237CHEBI:136235CHEBI:136489CHEBI:74372CHEBI:74371CHEBI:74241CHEBI:136239CHEBI:91039CHEBI:74375CHEBI:74374