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Mass spectrometry imaging (MSI)‐based spatially resolved metabolomics addresses the limitations inherent in traditional liquid chromatography‐tandem mass spectrometry (LC–MS)‐based metabolomics, particularly the loss of spatial context within heterogeneous tissues. MSI not only enhances our understanding of disease aetiology but also aids in the identification of biomarkers and the assessment of drug toxicity and therapeutic efficacy by converting invisible metabolites and biological networks into visually rendered image data. In this comprehensive review, we illuminate the key advancements in MSI‐driven spatially resolved metabolomics over the past few years. We first outline recent innovations in preprocessing methodologies and MSI instrumentation that improve the sensitivity and comprehensiveness of metabolite detection. We then delve into the progress made in functional visualization techniques, which enhance the precision of metabolite identification and annotation. Ultimately, we discuss the significant potential applications of spatially resolved metabolomics technology in translational medicine and drug development, offering new perspectives for future research and clinical translation. Highlights: MSI‐driven spatial metabolomics preserves metabolite spatial information, enhancing disease analysis and biomarker discovery.Advances in MSI technology improve detection sensitivity and accuracy, expanding bioanalytical applications.Enhanced visualization techniques refine metabolite identification and spatial distribution analysis.Integration of MSI with AI promises to advance precision medicine and accelerate drug development. [ABSTRACT FROM AUTHOR]

Surface‐assisted laser desorption/ionization (SALDI) mass spectrometry imaging (MSI) holds great value in spatial metabolomics and tumor diagnosis. Tissue imprinting on the SALDI target can avoid laser‐induced tissue ablation and simplifies the sample preparation. However, the tissue imprinting process always causes lateral diffusion of biomolecules, thereby losing the fidelity of metabolite distribution on tissue. Herein, a membrane‐mediated imprinting mass spectrometry imaging (MMI‐MSI) strategy is proposed using isoporous nuclepore track‐etched membrane as a mediating imprinting layer to selectively transport metabolites through uniform and vertical pores onto silicon nanowires (SiNWs) array. Compared with conventional direct imprinting technique, MMI‐MSI can not only exclude the adsorption of large biomolecules but also avoid the lateral diffusion of metabolites. The whole time for MMI‐based sample preparation can be reduced to 2 min, and the lipid peak number can increase from 46 to 113 in kidney tissue detection. Meanwhile, higher resolution of MSI can be achieved due to the confinement effect of the pore channel in the diffusion of metabolites. Based on MMI‐MSI, the tumor margins of liver cancer can be clearly discriminated and their different subtypes can be precisely classified. This work demonstrates MMI‐MSI is a rapid, highly sensitive, robust and high‐resolution technique for spatially‐resolved metabolomics and pathological diagnosis. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis (PF) is a chronic progressive end-stage lung disease. However, the mechanisms underlying the progression of this disease remain elusive. Presently, clinically employed drugs are scarce for the treatment of PF. Hence, there is an urgent need for developing novel drugs to address such diseases. Our study found for the first time that a natural source of Prismatomeris connata Y. Z. Ruan (Huang Gen, HG) ethyl acetate extract (HG-2) had a significant anti-PF effect by inhibiting the expression of the transforming growth factor beta 1/suppressor of mothers against decapentaplegic (TGF-β1/Smad) pathway. Network pharmacological analysis suggested that HG-2 had effects on tyrosine kinase phosphorylation, cellular response to reactive oxygen species, and extracellular matrix (ECM) disassembly. Moreover, mass spectrometry imaging (MSI) was used to visualize the heterogeneous distribution of endogenous metabolites in lung tissue and reveal the anti-PF metabolic mechanism of HG-2, which was related to arginine biosynthesis and alanine, asparate and glutamate metabolism, the downregulation of arachidonic acid metabolism, and the upregulation of glycerophospholipid metabolism. In conclusion, we elaborated on the relationship between metabolite distribution and the progression of PF, constructed the regulatory metabolic network of HG-2, and discovered the multi-target therapeutic effect of HG-2, which might be conducive to the development of new drugs for PF.

Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy. However, metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity. Herein, choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types, and a choline-modified strategy for small molecule-drug conjugates (SMDCs) design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy, instead of directly inhibiting choline metabolism. As a proof-of-concept, choline-modified SMDCs were designed, screened, and investigated for their druggability in vitro and in vivo. This strategy improved tumor targeting, preserved tumor inhibition and reduced toxicity of paclitaxel, through targeted drug delivery to tumor by highly expressed choline transporters, and site-specific release by carboxylesterase. This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy.

Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy. However, metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity. Herein, choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types, and a choline-modified strategy for small molecule-drug conjugates (SMDCs) design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy, instead of directly inhibiting choline metabolism. As a proof-of-concept, choline-modified SMDCs were designed, screened, and investigated for their druggability in vitro and in vivo. This strategy improved tumor targeting, preserved tumor inhibition and reduced toxicity of paclitaxel, through targeted drug delivery to tumor by highly expressed choline transporters, and site-specific release by carboxylesterase. This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy. [Display omitted] • Upregulated choline metabolism was discovered in multiple cancers by spatially resolved metabolomics. • Enhanced choline uptake was used as metabolic vulnerability for tumor-targeted therapy. • A choline-modified strategy to design small molecule-drug conjugate with anti-tumor activity and low toxicity. [ABSTRACT FROM AUTHOR]

Understanding the renal region-specific metabolic alteration in different animal models of diabetic nephropathy (DN) is critical for uncovering the underlying mechanisms and for developing effective treatments. In the present study, spatially resolved metabolomics based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was used to compare the local metabolic changes in the kidneys of HFD/STZ-induced diabetic rats and db/db mice. As a result, a total of 67 and 59 discriminating metabolites were identified and visualized in the kidneys of the HFD/STZ-induced diabetic rats and db/db mice, respectively. The result showed that there were significant region-specific changes in the glycolysis, TCA cycle, lipid metabolism, carnitine metabolism, choline metabolism, and purine metabolism in both DN models. However, the regional levels of the ten metabolites, including glucose, AMP, eicosenoic acid, eicosapentaenoic acid, Phosphatidylserine (36:1), Phosphatidylserine (36:4), Phosphatidylethanolamine (34:1), Phosphatidylethanolamine (36:4), Phosphatidylcholine (34:2), Phosphatidylinositol (38:5) were changed in reversed directions, indicating significant differences in the local metabolic phenotypes of these two commonly used DN animal models. This study provides comprehensive and in-depth analysis of the differences in the tissue and molecular pathological features in diabetic kidney injury in HFD/STZ-induced diabetic rats and db/db mice. [ABSTRACT FROM AUTHOR]

Understanding the renal region-specific metabolic alteration in different animal models of diabetic nephropathy (DN) is critical for uncovering the underlying mechanisms and for developing effective treatments. In the present study, spatially resolved metabolomics based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was used to compare the local metabolic changes in the kidneys of HFD/STZ-induced diabetic rats and db/db mice. As a result, a total of 67 and 59 discriminating metabolites were identified and visualized in the kidneys of the HFD/STZ-induced diabetic rats and db/db mice, respectively. The result showed that there were significant region-specific changes in the glycolysis, TCA cycle, lipid metabolism, carnitine metabolism, choline metabolism, and purine metabolism in both DN models. However, the regional levels of the ten metabolites, including glucose, AMP, eicosenoic acid, eicosapentaenoic acid, Phosphatidylserine (36:1), Phosphatidylserine (36:4), Phosphatidylethanolamine (34:1), Phosphatidylethanolamine (36:4), Phosphatidylcholine (34:2), Phosphatidylinositol (38:5) were changed in reversed directions, indicating significant differences in the local metabolic phenotypes of these two commonly used DN animal models. This study provides comprehensive and in-depth analysis of the differences in the tissue and molecular pathological features in diabetic kidney injury in HFD/STZ-induced diabetic rats and db/db mice.

Pulmonary fibrosis (PF) is a chronic progressive end-stage lung disease. However, the mechanisms underlying the progression of this disease remain elusive. Presently, clinically employed drugs are scarce for the treatment of PF. Hence, there is an urgent need for developing novel drugs to address such diseases. Our study found for the first time that a natural source of Prismatomeris connata Y. Z. Ruan (Huang Gen, HG) ethyl acetate extract (HG-2) had a significant anti-PF effect by inhibiting the expression of the transforming growth factor beta 1/suppressor of mothers against decapentaplegic (TGF-β1/Smad) pathway. Network pharmacological analysis suggested that HG-2 had effects on tyrosine kinase phosphorylation, cellular response to reactive oxygen species, and extracellular matrix (ECM) disassembly. Moreover, mass spectrometry imaging (MSI) was used to visualize the heterogeneous distribution of endogenous metabolites in lung tissue and reveal the anti-PF metabolic mechanism of HG-2, which was related to arginine biosynthesis and alanine, asparate and glutamate metabolism, the downregulation of arachidonic acid metabolism, and the upregulation of glycerophospholipid metabolism. In conclusion, we elaborated on the relationship between metabolite distribution and the progression of PF, constructed the regulatory metabolic network of HG-2, and discovered the multi-target therapeutic effect of HG-2, which might be conducive to the development of new drugs for PF. [Display omitted] • HG-2 can improve pulmonary fibrosis by inhibiting the TGF-β1/Smad pathway. • HG-2 can ameliorate the metabolic disorders of pulmonary fibrosis in mice. • MSI reveals the heterogeneous distribution of metabolites in pulmonary fibrosis. • HG-2 is expected to be developed as a multi-target drug for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Spatially resolved metabolomics is an excellent tool for elucidating in situ molecular events, but its use remains challenging due to the complexity of the endogenous metabolites in bio-tissue and tissue heterogeneity. In this study, a data processing pipeline for spatially resolved metabolomics analysis of tumor microregion heterogeneity was developed and built into a graphical interface with MSI software. Biological tissue sections were analysed by ambient air-flow assisted desorption electrospray ionization mass spectrometry imaging. Histology-driven and characterized ion images overlay combined with metabolic feature-based spatial segmentation were developed to accurately extract the metabolic profile from the tissue microregion of interest. In addition, appropriate data pretreatment methods were investigated to evaluate their ability to identify biological variations from the complicated spatially resolved metabolomics data. Diverse graphical metabolic feature extraction and various data pretreatment methods enable not only the achievement of the best multivariate statistical results in an intuitive and simple way but also the discovery of low-abundance but reliable biomarkers. The results from a papillary thyroid cancer tissue study demonstrated that this data processing pipeline is a powerful and easy-to-use tool for investigating the spatial molecular events in tumor microenvironments and to therefore thoroughly understand their metabolic heterogeneity. Image 1 • Providing a graphical and easy-to-use data processing pipeline for spatially resolved metabolomics analysis. • Metabolic feature based spatial segmentation to automatically present tissue heterogeneity. • Highlights biological variations to discover low-abundance but reliable in situ biomarkers. [ABSTRACT FROM AUTHOR]

P. lobata and P. thomsonii are medicinal plants with similar pharmacological functions but different therapeutic effects. A novel method is presented herein to investigate metabolites in terms of their distribution and qualification, quantification is necessary to elucidate the different therapeutic effects of the two Puerariae species. The aim of the present study was to perform spatially resolved metabolomics combined with bioactivity analyses to systematically compare the metabolite differences in P. lobata and P. thomsonii by distribution, qualification, quantification, and biological activity to evaluate their pharmacological properties. Air flow-assisted desorption electrospray ionization–mass spectrometry imaging (AFADESI-MSI) was performed to characterize the differences in the metabolite distributions of P. lobata and P. thomsonii. Further qualitative and quantitative analyses of the differential metabolites were performed using liquid chromatography–mass spectrometry (LC-MS). Biological activities correlated with the differences in the metabolites were validated by MTT assays. Some metabolites showed complementary distributions of the phloem and xylem in the two species, saccharide, vitamin, and inosine levels were higher in the phloem of P. thomsonii but higher in the xylem of P. lobata. The 3′-hydroxyl puerarin level was higher in the xylem of P. thomsonii but higher in the phloem of P. lobata. Qualitative and quantitative analyses of the metabolites revealed a total of 52 key differential metabolites. MTT assays showed that daidzein, daidzin, puerarin, ononin, genistin, formononetin, 3′-hydroxy puerarin, 3′-methoxy puerarin, mirificin, and 3′-methoxy daidzin exerted protective effects on H9c2 cells against hypoxia/reoxygenation injury. P. lobata extracts exhibited a significantly better protective efficacy than P. thomsonii extracts. In this study, AFADESI-MSI combined with LC-MS and biological activities comprehensively elucidated metabolite differences in the distribution, qualification, quantification, and pharmacological properties of P. lobata and P. thomsonii. The results of this study could provide a novel strategy for species identification and quality assessment of similar Chinese herbal medicines. [Display omitted] • AFADESI-MSI enables wide-coverage detection of massive metabolites in Puerariae. • The heterogeneous spatial features of metabolites in Pueraria were imaged. • Distinguishing two Puerariae species with AFADESI-MSI and biological activities. [ABSTRACT FROM AUTHOR]

The intricate physiological and pathological activities of the central nervous system (CNS) entail the translocation and regulation of a diverse set of biochemicals. Metabolomics is employed for the detection and annotation of metabolic events involved in biological pathways, allowing a better understanding of systems biology. Spatially resolved metabolomics has been recently developed to determine the association between the spatial distribution of metabolites and complex functional microregions of heterogeneous tissues. Due to its unique advantages, spatially resolved metabolomics is being increasingly employed in CNS research. This review discusses the principles and processes of spatially resolved metabolomics, focusing on some of the latest technological advances and its emerging application in the CNS field and summarizes our current understanding of this developing area, in an attempt to guide future research on this topic. • Spatially resolved metabolomics for central nervous system research. • Techniques to monitor spatial distribution and content changes in CNS metabolites. • Key parameters and impact factors in spatially resolved metabolomics. • Metabolic networks across functional brain tissue microregions. [ABSTRACT FROM AUTHOR]

The liver toxicity of Reynoutria multiflora (Thunb.) Moldenke. (Polygonaceae) (Polygonum multiflorum Thunb, PM) has always attracted much attention, but the related toxicity materials and mechanisms have not been elucidated due to multi-component and multi-target characteristics. In previous hepatotoxicity screening, different components of PM were first evaluated and the hepatotoxicity of component D [95% ethanol (EtOH) elution] in a 70% EtOH extract of PM (PM-D) showed the highest hepatotoxicity. Furthermore, the main components of PM-D were identified and their hepatotoxicity was evaluated based on a zebrafish embryo model. However, the hepatotoxicity mechanism of PM-D is unknown. This work is to explore the hepatotoxicity mechanisms of PM-D by integrating network toxicology and spatially resolved metabolomics strategy. A hepatotoxicity interaction network of PM-D was constructed based on toxicity target prediction for eight key toxic ingredients and a hepatotoxicity target collection. Then the key signaling pathways were enriched, and molecular docking verification was implemented to evaluate the ability of toxic ingredients to bind to the core targets. The pathological changes of liver tissues and serum biochemical assays of mice were used to evaluate the liver injury effect of mice with oral administration of PM-D. Furthermore, spatially resolved metabolomics was used to visualize significant differences in metabolic profiles in mice after drug administration, to screen hepatotoxicity-related biomarkers and analyze metabolic pathways. The contents of four key toxic compounds in PM-D were detected. Network toxicology identified 30 potential targets of liver toxicity of PM-D. GO and KEGG enrichment analyses indicated that the hepatotoxicity of PM-D involved multiple biological activities, including cellular response to endogenous stimulus, organonitrogen compound metabolic process, regulation of the apoptotic process, regulation of kinase, regulation of reactive oxygen species metabolic process and signaling pathways including PI3K-Akt, AMPK, MAPK, mTOR, Ras and HIF-1. The molecular docking confirmed the high binding activity of 8 key toxic ingredients with 10 core targets, including mTOR, PIK3CA, AKT1, and EGFR. The high distribution of metabolites of PM-D in the liver of administrated mice was recognized by mass spectrometry imaging. Spatially resolved metabolomics results revealed significant changes in metabolic profiles after PM-D administration, and metabolites such as taurine, taurocholic acid, adenosine, and acyl-carnitines were associated with PM-D-induced liver injury. Enrichment analyses of metabolic pathways revealed tht linolenic acid and linoleic acid metabolism, carnitine synthesis, oxidation of branched-chain fatty acids, and six other metabolic pathways were significantly changed. Comprehensive analysis revealed that the hepatotoxicity caused by PM-D was closely related to cholestasis, mitochondrial damage, oxidative stress and energy metabolism, and lipid metabolism disorders. In this study, the hepatotoxicity mechanisms of PM-D were comprehensively identified through an integrated spatially resolved metabolomics and network toxicology strategy, providing a theoretical foundation for the toxicity mechanisms of PM and its safe clinical application. [Display omitted] • The contents of key ingredients in PM-D were quantified. • Network toxicology was an effective tool for toxic mechanism of TCMs. • The toxic metabolic mechanism of PM-D was visualized by MSI technology. • An integrated strategy was proposed for investigating the toxicity mechanism of TCMs. [ABSTRACT FROM AUTHOR]

Spatially resolved metabolomics offers unprecedented opportunities for elucidating metabolic mechanisms during cancer progression. It facilitated the discovery of aberrant cellular metabolism with clinical application potential. Here, we developed a novel strategy to discover cancer tissue relevant metabolic signatures by high spatially resolved metabolomics combined with a multicellular tumor spheroid (MCTS) in vitro model. Esophageal cancer MCTS were generated using KYSE-30 human esophageal cancer cells to fully mimic the 3D microenvironment under physiological conditions. Then, the spatial features and temporal variation of metabolites and metabolic pathways in MCTS were accurately mapped by using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with a spatial resolution at ∼12 μm. Metabolites, such as glutamate, tyrosine, inosine and various types of lipids displayed heterogeneous distributions in different microregions inside the MCTS, revealing the metabolic heterogenicity of cancer cells under different proliferative states. Subsequently, through joint analysis of metabolomic data of clinical cancer tissue samples, cancer tissue relevant metabolic signatures in esophageal cancer MCTS were identified, including glutamine metabolism, fatty acid metabolism, de novo synthesis phosphatidylcholine (PC) and phosphatidylethanolamine (PE), etc. In addition, the abnormal expression of the involved metabolic enzymes, i.e., GLS, FASN, CHKA and cPLA2, was further confirmed and showed similar tendencies in esophageal cancer MCTS and cancer tissues. The MALDI-MSI combined with MCTS approach offers molecular insights into cancer metabolism with real-word relevance, which would potentially benefit the biomarker discovery and metabolic mechanism studies. [Display omitted] • A high spatially resolved metabolomics method allows to map metabolite distributions within ∼1 mm diameter MCTS. • The heterogeneous spatial features and dynamics of metabolites in esophageal cancer MCTS were successfully characterized. • Esophageal cancer MCTS displayed a good similarity with clinical esophageal cancer tissues at metabolite and metabolic enzyme levels. • The combination of MCTS and MALDI-MSI provides a good strategy to discover cancer tissue relevant metabolic signatures. [ABSTRACT FROM AUTHOR]