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Background: Metabolic reprogramming is a common phenomenon in tumorigenesis and tumor progression. Amino acids are important mediators in cancer metabolism, and their kinetics in tumor tissue are far from being understood completely. Mass spectrometry imaging is capable to spatiotemporally trace important endogenous metabolites in biological tissue specimens. In this research, we studied L-[ring- 13 C 6 ]-labeled phenylalanine and tyrosine kinetics in a human non-small cell lung carcinoma (NSCLC) xenografted mouse model using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI)., Methods: We investigated the L-[ring- 13 C 6 ]-Phenylalanine ( 13 C 6 -Phe) and L-[ring- 13 C 6 ]-Tyrosine ( 13 C 6 -Tyr) kinetics at 10 min (n = 4), 30 min (n = 3), and 60 min (n = 4) after tracer injection and sham-treated group (n = 3) at 10 min in mouse-xenograft lung tumor tissues by MALDI-FTICR-MSI., Results: The dynamic changes in the spatial distributions of 19 out of 20 standard amino acids are observed in the tumor tissue. The highest abundance of 13 C 6 -Phe was detected in tumor tissue at 10 min after tracer injection and decreased progressively over time. The overall enrichment of 13 C 6 -Tyr showed a delayed temporal trend compared to 13 C 6 -Phe in tumor caused by the Phe-to-Tyr conversion process. Specifically, 13 C 6 -Phe and 13 C 6 -Tyr showed higher abundances in viable tumor regions compared to non-viable regions., Conclusions: We demonstrated the spatiotemporal intra-tumoral distribution of the essential aromatic amino acid 13 C 6 -Phe and its de-novo synthesized metabolite 13 C 6 -Tyr by MALDI-FTICR-MSI. Our results explore for the first time local phenylalanine metabolism in the context of cancer tissue morphology. This opens a new way to understand amino acid metabolism within the tumor and its microenvironment.

In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis., (© 2020 Rocha et al.)

Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As part of a project to demonstrate the science of decay, a series of mass spectrometry imaging experiments were performed. The aim was to demonstrate that decay and decomposition are only part of the story and to show pictorially that atoms and molecules from dead plants and animals are incorporated into new life. Radish plants (Raphanus sativus) were grown hydroponically using a nutrient system containing (15)N KNO3 (98% labeled) as the only source of nitrogen. Plants were cropped and left to ferment in water for 2 weeks to create a radish "tea", which was used as a source of nitrogen for radish grown in a second hydroponics experiment. After 5 weeks of growth, the radish plants were harvested and cryosectioned, and sections were imaged by positive-ion MALDI and SIMS mass spectrometry imaging. The presence of labeled species in the plants grown using (15)N KNO3 as nutrient and those grown from the radish "tea" was readily discernible. The uptake of (15)N into a number of identifiable metabolites has been studied by MALDI-MS and SIMS imaging.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Cardiovascular diseases are the world's number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na(+), K(+), and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

Osteoarthritis (OA) is a pathology that ultimately causes joint destruction. The cartilage is one of the principal affected tissues. Alterations in the lipid mediators and an imbalance in the metabolism of cells that form the cartilage (chondrocytes) have been described as contributors to the OA development. In this study, we have studied the distribution of lipids and chemical elements in healthy and OA human cartilage. Time of flight-secondary ion mass spectrometry (TOF-SIMS) allows us to study the spatial distribution of molecules at a high resolution on a tissue section. TOF-SIMS revealed a specific peak profile that distinguishes healthy from OA cartilages. The spatial distribution of cholesterol-related peaks exhibited a remarkable difference between healthy and OA cartilages. A distinctive colocalization of cholesterol and other lipids in the superficial area of the cartilage was found. A higher intensity of oleic acid and other fatty acids in the OA cartilages exhibited a similar localization. On the other hand, CN(-) was observed with a higher intensity in the healthy samples. Finally, we observed an accumulation of calcium and phosphate ions exclusively in areas surrounding the chondrocyte in OA tissues. To our knowledge, this is the first time that TOF-SIMS revealed combined changes in the molecular distribution in the OA human cartilage.