Next‐generation imaging mass spectrometry of biological tissue on the cellular level shows excellent agreement with histochemical evaluation whilst providing a much higher discrimination capacity based on label‐free molecular information (see image of a mouse urinary bladder section).

BackgroundSoluble protein and lipid mediators play essential roles in the tumor environment, but their cellular origins, targets, and clinical relevance are only partially known. We have addressed this question for the most abundant cell types in human ovarian carcinoma ascites, namely tumor cells and tumor-associated macrophages.ResultsTranscriptome-derived datasets were adjusted for errors caused by contaminating cell types by an algorithm using expression data derived from pure cell types as references. These data were utilized to construct a network of autocrine and paracrine signaling pathways comprising 358 common and 58 patient-specific signaling mediators and their receptors. RNA sequencing based predictions were confirmed for several proteins and lipid mediators. Published expression microarray results for 1018 patients were used to establish clinical correlations for a number of components with distinct cellular origins and target cells. Clear associations with early relapse were found for STAT3-inducing cytokines, specific components of WNT and fibroblast growth factor signaling, ephrin and semaphorin axon guidance molecules, and TGFβ/BMP-triggered pathways. An association with early relapse was also observed for secretory macrophage-derived phospholipase PLA2G7, its product arachidonic acid (AA) and signaling pathways controlled by the AA metabolites PGE2, PGI2, and LTB4. By contrast, the genes encoding norrin and its receptor frizzled 4, both selectively expressed by cancer cells and previously not linked to tumor suppression, show a striking association with a favorable clinical course.ConclusionsWe have established a signaling network operating in the ovarian cancer microenvironment with previously unidentified pathways and have defined clinically relevant components within this network.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0956-6) contains supplementary material, which is available to authorized users.

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100,000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.

The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.

Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of ω-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators.