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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]

Metabolic abnormalities in mammalian joint tissues are closely linked to various diseases, including rheumatoid arthritis and osteoarthritis. The variations of small molecule metabolites within bone joint tissues can affect cell proliferation, drive the secretion of inflammatory mediators, mediate leukocyte infiltration, and consequently induce synovial inflammation and cartilage damage. Therefore, the development of novel mass spectrometry imaging (MSI) methods for visualizing metabolites in joint tissues is crucial for comprehensively understanding the metabolic characteristics of different microregions under both physiological and pathological conditions. In this study, an airflow-assisted desorption electrospray ionization (AFADESI)-MSI was used to establish an ambient MSI method for rat joint tissues. To ensure the effectiveness of the method, dynamic range, sensitivity, and imaging effect were selected as the primary evaluation criteria. The tissue preparation, section thickness, and spray solvent systems were systematically optimized. Compared to the AB glue transfer method, the tape adhesion method can obtain complete bone joint tissue sections and clearer images. A section thickness of 20 μm, as opposed to 15 μm, provides higher ion intensity in the m/z 600-900 range. Among the tested spray solvent systems, compared with methanol-water (8:2, V/V), acetonitrile-water (8:2, V/V), and the acetonitrile-isopropanol-water (6:2:2, V/V/V), the acetonitrile-isopropanol-water (4:4:2, V/V/V) prove to be the most effective, detecting the highest number of metabolites and lipids when employed as the spray solvent. The precision of this method was evaluated on six adjacent rat knee sections under positive and negative ion modes, with the relative standard deviation (RSD) for the ion intensity of representative metabolites being less than 20%. Subsequently, this method was utilized for spatially resolved metabolomics analysis of rat knee tissues. A total of 613 metabolites are annotated in the overall knee tissues. Based on the optical images, the rat knee tissues were segmented into three microregions for bone, marrow, and cartilage. 452, 418, and 451 metabolites in these microregions are identified, respectively. In bone, the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and the TCA cycle are more prominent. In bone marrow, ascorbate and purine metabolism pathways are more significant. In cartilage, arginine and proline metabolism, as well as lysine degradation, are more pronounced. These findings highlight significant metabolic heterogeneity across different microregions of rat knee joint tissues. Overall, this study develops an AFADESI-MSI method for in-depth profiling of metabolites within rat joint tissues, providing a powerful tool for in situ visualization of metabolites in metabolic studies of joint tissues. [ABSTRACT FROM AUTHOR]

Lipid metabolism is altered in rapidly proliferating cancer cells, where fatty acids (FAs) are utilized in the synthesis of sphingolipids and glycerophospholipids to produce cell membranes and signaling molecules. Receptor for activated C‐kinase 1 (RACK1; also known as small ribosomal subunit protein) is an intracellular scaffolding protein involved in signaling pathways. Whether such lipid metabolism is regulated by RACK1 is unknown. Here, integrated spatially resolved metabolomics and spatial transcriptomics revealed that accumulation of lipids in cervical cancer (CC) samples correlated with overexpression of RACK1, and RACK1 promoted lipid synthesis in CC cells. Chromatin immunoprecipitation verified binding of sterol regulatory element‐binding protein 1 (SREBP1) to acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FASN) promoters. RACK1 enhanced de novo FA synthesis by upregulating expression of sterol regulatory element binding transcription factor 1 (SREBP1) and lipogenic genes FASN and ACC1. Co‐immunoprecipitation and western blotting revealed that RACK1 interacted with protein kinase B (AKT) to activate the AKT/mammalian target of rapamycin (mTOR)/SREBP1 signaling pathway to promote FA synthesis. Cell proliferation and apoptosis experiments suggested that RACK1‐regulated FA synthesis is key in the progression of CC. Thus, RACK1 enhanced lipid synthesis through the AKT/mTOR/SREBP1 signaling pathway to promote the growth of CC cells. RACK1 may become a therapeutic target for CC. [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]

Metabolic abnormalities in mammalian joint tissues are closely linked to various diseases, including rheumatoid arthritis and osteoarthritis. The variations of small molecule metabolites within bone joint tissues can affect cell proliferation, drive the secretion of inflammatory mediators, mediate leukocyte infiltration, and consequently induce synovial inflammation and cartilage damage. Therefore, the development of novel mass spectrometry imaging (MSI) methods for visualizing metabolites in joint tissues is crucial for comprehensively understanding the metabolic characteristics of different microregions under both physiological and pathological conditions. In this study, an airflow-assisted desorption electrospray ionization (AFADESI)-MSI was used to establish an ambient MSI method for rat joint tissues. To ensure the effectiveness of the method, dynamic range, sensitivity, and imaging effect were selected as the primary evaluation criteria. The tissue preparation, section thickness, and spray solvent systems were systematically optimized. Compared to the AB glue transfer method, the tape adhesion method can obtain complete bone joint tissue sections and clearer images. A section thickness of 20 μm, as opposed to 15 μm, provides higher ion intensity in the m/z 600-900 range. Among the tested spray solvent systems, compared with methanol-water (8:2, V/V), acetonitrile-water (8:2, V/V), and the acetonitrile-isopropanol-water (6:2:2, V/V/V), the acetonitrile-isopropanol-water (4:4:2, V/V/V) prove to be the most effective, detecting the highest number of metabolites and lipids when employed as the spray solvent. The precision of this method was evaluated on six adjacent rat knee sections under positive and negative ion modes, with the relative standard deviation (RSD) for the ion intensity of representative metabolites being less than 20%. Subsequently, this method was utilized for spatially resolved metabolomics analysis of rat knee tissues. A total of 613 metabolites are annotated in the overall knee tissues. Based on the optical images, the rat knee tissues were segmented into three microregions for bone, marrow, and cartilage. 452, 418, and 451 metabolites in these microregions are identified, respectively. In bone, the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and the TCA cycle are more prominent. In bone marrow, ascorbate and purine metabolism pathways are more significant. In cartilage, arginine and proline metabolism, as well as lysine degradation, are more pronounced. These findings highlight significant metabolic heterogeneity across different microregions of rat knee joint tissues. Overall, this study develops an AFADESI-MSI method for in-depth profiling of metabolites within rat joint tissues, providing a powerful tool for in situ visualization of metabolites in metabolic studies of joint tissues.

Abstract 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.

High‐coverage mass spectrometry analysis of single‐cell metabolomics remains challenging due to the extremely low abundance and wide polarity of metabolites and ultra‐small volume in single cells. Herein, a novel concentric hybrid ionization source, nanoelectrospray ionization‐atmospheric pressure chemical ionization (nanoESI‐APCI), is ingeniously designed to detect polar and nonpolar metabolites simultaneously in single cells. The source is constructed by inserting a pulled glass capillary coaxially into a glass tube that acts as a dielectric barrier layer. Benefitting from the integrated advantages of nanoESI and APCI, its limit of detection is improved by one order of magnitude to 10 pg mL−1. After the operational parameter optimization, 254 metabolites detected in nanoESI‐APCI are tentatively identified from a single cell, and 82 more than those in nanoESI. The developed nanoESI‐APCI is successively applied to study the metabolic heterogeneity of human hepatocellular carcinoma tissue microenvironment united with laser capture microdissection (LCM), the discrimination of cancer cell types and subtypes, the metabolic perturbations to glucose starvation in MCF7 cells and the metabolic regulation of cancer stem cells. These results demonstrated that the nanoESI‐APCI not only opens a new avenue for high‐coverage and high‐sensitivity metabolomics analysis of single cell, but also facilitates spatially resolved metabolomics study coupled with LCM. [ABSTRACT FROM AUTHOR]

Casper, a type of transparent mutant-line zebrafish, was generated to overcome the opaque trunk of an adult zebrafish for tumor modeling to realize real-time visualization of transplanted cells in vivo. However, the molecular information at the metabolic level has not received much attention. Herein, a spatially resolved metabolomics method based on an airflow-assisted desorption electrospray ionization–mass spectrometry imaging (AFADESI-MSI) system for whole-body zebrafish was used to investigate small molecules and the distribution of adult casper (Mitfaw2/w2, roya9/a9) and the differences from wild-type zebrafish. Finally, the spatial distribution information of more than 1500 endogenous ions was obtained in positive and negative detection modes, and 186 metabolites belonging to a variety of structural categories were identified or annotated. Compared with wild-type samples, 85 variables, including 37 known metabolites, were screened out. In addition, the disordered metabolic pathways caused by the genetic mutation were excavated, involving downregulation of purine metabolism and arachidonic acid metabolism, upregulation of glycerophospholipid metabolism, and biosynthesis of unsaturated fatty acids. All these results were observed in the most intuitive way through MSI. This study revealed important metabolic characteristics of and perturbation in adult casper zebrafish, and provides indispensable fundamental knowledge for tumor research based on it. [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.

Abstract High‐coverage mass spectrometry analysis of single‐cell metabolomics remains challenging due to the extremely low abundance and wide polarity of metabolites and ultra‐small volume in single cells. Herein, a novel concentric hybrid ionization source, nanoelectrospray ionization‐atmospheric pressure chemical ionization (nanoESI‐APCI), is ingeniously designed to detect polar and nonpolar metabolites simultaneously in single cells. The source is constructed by inserting a pulled glass capillary coaxially into a glass tube that acts as a dielectric barrier layer. Benefitting from the integrated advantages of nanoESI and APCI, its limit of detection is improved by one order of magnitude to 10 pg mL−1. After the operational parameter optimization, 254 metabolites detected in nanoESI‐APCI are tentatively identified from a single cell, and 82 more than those in nanoESI. The developed nanoESI‐APCI is successively applied to study the metabolic heterogeneity of human hepatocellular carcinoma tissue microenvironment united with laser capture microdissection (LCM), the discrimination of cancer cell types and subtypes, the metabolic perturbations to glucose starvation in MCF7 cells and the metabolic regulation of cancer stem cells. These results demonstrated that the nanoESI‐APCI not only opens a new avenue for high‐coverage and high‐sensitivity metabolomics analysis of single cell, but also facilitates spatially resolved metabolomics study coupled with LCM.

Tumors are spatially heterogeneous tissues that comprise numerous cell types with intricate structures. By interacting with the microenvironment, tumor cells undergo dynamic changes in gene expression and metabolism, resulting in spatiotemporal variations in their capacity for proliferation and metastasis. In recent years, the rapid development of histological techniques has enabled efficient and high-throughput biomolecule analysis. By preserving location information while obtaining a large number of gene and molecular data, spatially resolved metabolomics (SRM) and spatially resolved transcriptomics (SRT) approaches can offer new ideas and reliable tools for the in-depth study of tumors. This review provides a comprehensive introduction and summary of the fundamental principles and research methods used for SRM and SRT techniques, as well as a review of their applications in cancer-related fields.

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.

Casper, a type of transparent mutant-line zebrafish, was generated to overcome the opaque trunk of an adult zebrafish for tumor modeling to realize real-time visualization of transplanted cells in vivo. However, the molecular information at the metabolic level has not received much attention. Herein, a spatially resolved metabolomics method based on an airflow-assisted desorption electrospray ionization–mass spectrometry imaging (AFADESI-MSI) system for whole-body zebrafish was used to investigate small molecules and the distribution of adult casper (Mitfaw2/w2, roya9/a9) and the differences from wild-type zebrafish. Finally, the spatial distribution information of more than 1500 endogenous ions was obtained in positive and negative detection modes, and 186 metabolites belonging to a variety of structural categories were identified or annotated. Compared with wild-type samples, 85 variables, including 37 known metabolites, were screened out. In addition, the disordered metabolic pathways caused by the genetic mutation were excavated, involving downregulation of purine metabolism and arachidonic acid metabolism, upregulation of glycerophospholipid metabolism, and biosynthesis of unsaturated fatty acids. All these results were observed in the most intuitive way through MSI. This study revealed important metabolic characteristics of and perturbation in adult casper zebrafish, and provides indispensable fundamental knowledge for tumor research based on it.

ABSTRACTAlthough gut microbiota has been linked to cancer, little is known about the crosstalk between gut- and intratumoral-microbiomes. The goal of this study was to determine whether gut Akkermansia muciniphila (Akk) is involved in the regulation of intratumoral microbiome and metabolic contexture, leading to an anticancer effect on lung cancer. We evaluated the effects of gut endogenous or gavaged exogenous Akk on the tumorigenesis using the Lewis lung cancer mouse model. Feces, blood, and tumor tissue samples were collected for 16S rDNA sequencing. We then conducted spatially resolved metabolomics profiling to discover cancer metabolites in situ directly and to characterize the overall Akk-regulated metabolic features, followed by the correlation analysis of intratumoral bacteria with metabolic network. Our results showed that both endogenous and exogenous gavaged Akk significantly inhibited tumorigenesis. Moreover, we detected increased Akk abundance in blood circulation or tumor tissue by 16S rDNA sequencing in the Akk gavaged mice, compared with the control mice. Of great interest, gavaged Akk may migrate into tumor tissue and influence the composition of intratumoral microbiome. Spatially resolved metabolomics analysis revealed that the gut-derived Akk was able to regulate tumor metabolic pathways, from metabolites to enzymes. Finally, our study identified a significant correlation between the gut Akk-regulated intratumoral bacteria and metabolic network. Together, gut-derived Akk may migrate into blood circulation, and subsequently colonize into lung cancer tissue, which contributes to the suppression of tumorigenesis by influencing tumoral symbiotic microbiome and reprogramming tumoral metabolism, although more studies are needed.

Tumors are spatially heterogeneous tissues that comprise numerous cell types with intricate structures. By interacting with the microenvironment, tumor cells undergo dynamic changes in gene expression and metabolism, resulting in spatiotemporal variations in their capacity for proliferation and metastasis. In recent years, the rapid development of histological techniques has enabled efficient and high-throughput biomolecule analysis. By preserving location information while obtaining a large number of gene and molecular data, spatially resolved metabolomics (SRM) and spatially resolved transcriptomics (SRT) approaches can offer new ideas and reliable tools for the in-depth study of tumors. This review provides a comprehensive introduction and summary of the fundamental principles and research methods used for SRM and SRT techniques, as well as a review of their applications in cancer-related fields. [Display omitted] • The principles and characteristics of SRT and SRM techniques are summarized. • Tumors show spatiotemporal variation via microenvironment interactions. • Single-cell spatially resolved omics reveals tumor heterogeneity. [ABSTRACT FROM AUTHOR]

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]

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]

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.

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 response of wheat to elevated atmospheric CO 2 concentration (e [CO 2 ]) within a single generation is being cumulatively studied, but multigenerational effects of e [CO 2 ] on wheat is largely unknown. In this study, the emerging spatially resolved metabolomics was used to visualize the spatial distributions of amino acids and organic acids in wheat caryopsis exposed to multigenerational e [CO 2 ]. These results showed that nine kinds of amino acids and three kinds of organic acids exhibited embryo-distribution pattern in wheat caryopsis under sequential atmospheric CO 2 concentration (a [CO 2 ]). Additionally, e [CO 2 ] can induce nonuniformly spatial distribution of amino acids and organic acids. Further inspection of the differential accumulation of amino acids in embryo/endosperm under multigenerational e [CO 2 ] exposure indicated that the abundance of valine, glutamine, glutamic acid and histidine in embryo was decreased by multigenerational e [CO 2 ] exposure, whereas abundance of lysine, aspartic acid and glutamic acid in endosperm showed different trends. Multigenerational e [CO 2 ] exposure decreased the abundance of ascorbic acid and cis -aconitic acid, regardless of regions, while increased the abundance of malic acid in endosperm. It was suggested that the long-term e [CO 2 ] cause inconsistent changes in embryo and endosperm, which may lead to potential impacts on human dietary health and crop offspring vigor. [Display omitted] • The response of wheat to multigenerational effects of e [CO 2 ] had important effects. • This effects of multigenerational e [CO 2 ] displayed grain quality and offspring vigor. • Spatially resolved metabolomics provided a first snapshot of metabolites in caryopsis. • Spatial distribution of amino acids and organic acids played a major role. [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]

Although gut microbiota has been linked to cancer, little is known about the crosstalk between gut- and intratumoral-microbiomes. The goal of this study was to determine whether gut Akkermansia muciniphila (Akk) is involved in the regulation of intratumoral microbiome and metabolic contexture, leading to an anticancer effect on lung cancer. We evaluated the effects of gut endogenous or gavaged exogenous Akk on the tumorigenesis using the Lewis lung cancer mouse model. Feces, blood, and tumor tissue samples were collected for 16S rDNA sequencing. We then conducted spatially resolved metabolomics profiling to discover cancer metabolites in situ directly and to characterize the overall Akk-regulated metabolic features, followed by the correlation analysis of intratumoral bacteria with metabolic network. Our results showed that both endogenous and exogenous gavaged Akk significantly inhibited tumorigenesis. Moreover, we detected increased Akk abundance in blood circulation or tumor tissue by 16S rDNA sequencing in the Akk gavaged mice, compared with the control mice. Of great interest, gavaged Akk may migrate into tumor tissue and influence the composition of intratumoral microbiome. Spatially resolved metabolomics analysis revealed that the gut-derived Akk was able to regulate tumor metabolic pathways, from metabolites to enzymes. Finally, our study identified a significant correlation between the gut Akk-regulated intratumoral bacteria and metabolic network. Together, gut-derived Akk may migrate into blood circulation, and subsequently colonize into lung cancer tissue, which contributes to the suppression of tumorigenesis by influencing tumoral symbiotic microbiome and reprogramming tumoral metabolism, although more studies are needed.

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]

Spatially resolved metabolic profiling of brain is vital for elucidating tissue-specific molecular histology and pathology underlying diabetic encephalopathy (DE). In this study, a spatially resolved metabolomic method based on air-flow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) was developed for investigating the region-specific metabolic disturbances in the brain of DE model rats induced by a high-fat diet in combination with streptozotocin administration. A total of 19 discriminating metabolites associated with glycolysis and the pentose phosphate pathway (PPP); the glutamate/gamma aminobutyric acid-glutamine cycle and tricarboxylic acid cycle; nucleotide metabolism; lipid metabolism; carnitine homeostasis; and taurine, ascorbic acid, histidine, and choline metabolism were identified and located in the brains of the diabetic rats simultaneously for the first time. The results indicated that increased glycolytic and PPP activity; dysfunction of mitochondrial metabolism; dysregulation of adenosinergic, glutamatergic, dopaminergic, cholinergic, and histaminergic systems; disorder of osmotic regulation and antioxidant system; and disorder of lipid metabolism occur in a region-specific fashion in the brains of DE rats. Thus, this study provides valuable information regarding the molecular pathological signature of DE. These findings also underline the high potential of AFADESI-MSI for applications in various central nervous system diseases.