Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Article Title: Mass-Guided Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers.
doi: 10.1002/advs.202410506
Figure Lengend Snippet: Figure 4. MALDI MS imaging of hippocampal slice cultures suggests metabolic neuron-glia interplay in response to LPS-induced neuroinflammation. a) Rat hippocampal slice cultures following 72 h incubation with different treatments: vehicle-treated (VEH), 1 μg mL−1 lipopolysaccharide (LPS), or 100 μg mL−1 clodronate (CLO, a bisphosphonate for selective macrophage and microglia depletion).[29a,b] b) Volcano plot comparing VEH- and LPS- treated hippocampal slices revealed m/z features that mark non-activated and LPS-responding cells. Measurements were taken from n = 5 animals with 3 replicates per treatment, for a total of 30 sections. Benjamini Hochberg adjusted p-value threshold was set to 0.01 and Cohen´s D threshold to ± 0.2. Itaconate (m/z 129.02; [M-H]−) translates as a response marker from microglial cell populations (Figure 3) to slice cultures. c) Increased itaconate MSI ion intensities in LPS-treated hippocampal slices versus VEH and CLO controls (20 μm pixel size, negative ion mode). Anti-CD68-immunofluorescence overlay with Hoechst-stained nuclei suggests high microglia density in LPS-treated cultures. d) Metabolic profiling by MALDI MS imaging using KEGG METASPACE annotations at FDR<10%: Venn diagram comparing metabolites specific (Cohen´s D > 0.2 AND p < 0.01) for CLO slice cultures versus VEH&LPS cultures containing microglia. Then comparing LPS-treated slice culture against VEH. e) Comparison of slice culture with hiPSC and SIMA9 cell metabolite profiles, suggesting common markers of non-activated microglia: GABA*(m/z 102.06), fumarate (m/z 115.00), and glutamate (m/z 146.05) and of active microglia: Itaconate (m/z 129.02), ornithine (m/z 131.08) and hypoxanthine (m/z 135.03). The hiPSC profile shared non-activated markers N-acetyl-alanine (m/z = 130.05), adenine (m/z = 134.05), N-acetyl-aspartate (NAA) (m/z 174.04), FA 18:1 (m/z 281.25), and AMP (m/z 346.06) with slice cultures. f) Ion intensity of NAA is reduced in LPS-treated slice culture compared to VEH and CLO tissue. g) Hypothetical model of metabolic neuron- glia interplay in LPS-activated hippocampal slice cultures. In total five separate measurements were performed, with all three conditions measured in triplicates each time, resulting in 45 slice culture sections.
Article Snippet: Cell lines and Animal Tissues: SIMA9 mouse microglia cells (Cat. No. CRL-3265), EOC 13.31 mouse microglia cells (Cat. No. CRL-2468), and LADMAC macrophages (Cat. No. CRL-2420) were obtained from ATCC (Manassas, United States). hiPSC lines hiPSC1 (male) and hiPSC1 (female) were used for microglial differentiation.
Techniques: Imaging, Incubation, Marker, Staining, Comparison
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ATCC manufactures this product 
![Figure 1. PRISM-MS for fast low mass metabolite-preserving single-cell spatial metabolomics. a) Schematic overview of PRISM-MS workflow. (I) Survey PreScan at large (e.g., >100 μm) pixel size; (II) determine cell containing pixels by feature-selective binary image segmentation; (III) definition of cell containing pixels as new measurement regions; (IV) DeepScan at small (e.g., <20 μm) pixel size; followed by data analysis for single cell metabolomics and cluster detection (Figure S13, Supporting Information). b) PRISM-MS example of cultured <t>SIMA9</t> mouse microglia cells covered with MALDI matrix directly after lyophilization: the PreScan obtains cellular signal intensities per 200-μm pixels (green color scale) (upper panel left), which then get thresholded (upper panel middle) and undergo a subsequent DeepScan at 20-μm pixel size to resolve individual cells (upper panel right). Right dashed box: four distinct 200-μm measurement regions with 20 μm DeepScan step size (ion images of m/z 281.25 (FA 18:1 [M-H]−)). c) Left panel: 5-μm MSI image of cell marker m/z 281.25 (FA 18:1 [M-H]−); right panel: overlay of ion image with haematoxylin and eosin (H&E) -stained slide generated after MSI. The registration offset is deliberate, in order to demonstrate cell identification versus H&E ground truth. d) Intensity profiles for (i) m/z 279.23 (linoleic acid [M-H]−), (ii) m/z 306.08 (glutathione [M-H]−) and (iii) m/z 295.23 (9- and 13-hydroxy-octadecadienoic acids (9/13-HODE) [M-H]−) obtained by PRISM-MS (blue) versus the optically guided workflow (red). Oxidation of linoleic acid to 9/13-HODE is reduced in PRISM-MS. e) PRISM-MS preserves metabolite profiles: average (N = 3) Comparison of Cohen’s D effect sizes for small molecule m/z features obtained by PRISM-MS or by a workflow including 30 min slide exposure to ambient conditions to emulate workflows that capture, e.g., a high resolution optical image before MSI.[3c]](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_5230/pm39665230/pm39665230__page3_image1.jpg)
![Figure 3. Microglial responses to LPS treatment revealed sub-populations of SIMA9 and hiPSC microglia cells. a) Modified Volcano plot for untreated (vehicle; VEH) and lipopolysaccharide (LPS) treated (500 ng mL−1, 20 h) SIMA9 cells highlights m/z 124.01 (taurine [M-H]−) and m/z 129.02 (itaconate [M-H]−) as markers for non-activated and LPS-activated microglial cells, respectively. Benjamini- Hochberg adjusted p-value threshold was set to 0.05 and Cohen´s D threshold to ± 0.2. b) Itaconate violin plot: normalized intensity of itaconate per cell indicates microglial activation in correlation with LPS treatment. c) M3C cluster analysis of SIMA9 cells for itaconate and taurine labeling. Cells are categorized into four distinct groups by post analysis – itaconate-positive (Ita+Taur-, pink), taurine-positive (Ita-Taur+, green), and positive for both markers (Ita+Taur+, purple). Cells that don´t fall into either category (Ita-Taur-, grey) are omitted. d) Visualization of M3C cluster analysis for chamber slide wells untreated (VEH) or treated with a concentration range of LPS (0.1 to 500 ng mL−1). e) Besides murine SIMA9 cells, the hiPSC-derived microglia cell lines hiPSC1 and hiPSC2 cells responded to LPS- treatment, as indicated by itaconate increases. Murine EOC cells lack toll-like receptor 4 and did not respond to LPS. f) t-distributed stochastic neighbor embedding (t-SNE) for the four microglial cells lines. g) t-SNE analysis of microglial activation status indicates that only a subset of SIMA9, hiPSC1, and hiPSC2 cells were activated by LPS (500 ng mL−1 for 20 h): Cells were categorized as Ita-Taur+ (green), Ita+Taur+ (purple), Ita+Taur- (pink), or Ita-Taur- (grey). EOC cells were non-reactive. h) Volcano plot restricted to Ita-Taur+ and Ita+Taur- cells. Comparing both clusters yielded more defined m/z-signatures for microglial activation (15 markers of non-activation and 55 activation marker candidates) than using cell pools (2 markers of non- activation and 26 activation markers; compare a) by reducing extraneous data. In total 8.878 cells were analysed and measured in six separate runs.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_5230/pm39665230/pm39665230__page6_image1.jpg)
![Figure 4. MALDI MS imaging of hippocampal slice cultures suggests metabolic neuron-glia interplay in response to LPS-induced neuroinflammation. a) Rat hippocampal slice cultures following 72 h incubation with different treatments: vehicle-treated (VEH), 1 μg mL−1 lipopolysaccharide (LPS), or 100 μg mL−1 clodronate (CLO, a bisphosphonate for selective macrophage and microglia depletion).[29a,b] b) Volcano plot comparing VEH- and LPS- treated hippocampal slices revealed m/z features that mark non-activated and LPS-responding cells. Measurements were taken from n = 5 animals with 3 replicates per treatment, for a total of 30 sections. Benjamini Hochberg adjusted p-value threshold was set to 0.01 and Cohen´s D threshold to ± 0.2. Itaconate (m/z 129.02; [M-H]−) translates as a response marker from microglial cell populations (Figure 3) to slice cultures. c) Increased itaconate MSI ion intensities in LPS-treated hippocampal slices versus VEH and CLO controls (20 μm pixel size, negative ion mode). Anti-CD68-immunofluorescence overlay with Hoechst-stained nuclei suggests high microglia density in LPS-treated cultures. d) Metabolic profiling by MALDI MS imaging using KEGG METASPACE annotations at FDR<10%: Venn diagram comparing metabolites specific (Cohen´s D > 0.2 AND p < 0.01) for CLO slice cultures versus VEH&LPS cultures containing microglia. Then comparing LPS-treated slice culture against VEH. e) Comparison of slice culture with hiPSC and SIMA9 cell metabolite profiles, suggesting common markers of non-activated microglia: GABA*(m/z 102.06), fumarate (m/z 115.00), and glutamate (m/z 146.05) and of active microglia: Itaconate (m/z 129.02), ornithine (m/z 131.08) and hypoxanthine (m/z 135.03). The hiPSC profile shared non-activated markers N-acetyl-alanine (m/z = 130.05), adenine (m/z = 134.05), N-acetyl-aspartate (NAA) (m/z 174.04), FA 18:1 (m/z 281.25), and AMP (m/z 346.06) with slice cultures. f) Ion intensity of NAA is reduced in LPS-treated slice culture compared to VEH and CLO tissue. g) Hypothetical model of metabolic neuron- glia interplay in LPS-activated hippocampal slice cultures. In total five separate measurements were performed, with all three conditions measured in triplicates each time, resulting in 45 slice culture sections.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_5230/pm39665230/pm39665230__page8_image1.jpg)