Spatially resolved metabolomics visualizes heterogeneous distribution of metabolites in lung tissue and the anti-pulmonary fibrosis effect of Prismatomeris connate extract

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.

Spatiotemporal pharmacometabolomics based on ambient mass spectrometry imaging to evaluate the metabolism and hepatotoxicity of amiodarone in HepG2 spheroids

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and pathological conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of>1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in Ndesethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal information for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Discovering metabolic vulnerability using spatially resolved metabolomics for antitumor small molecule-drug conjugates development as a precise cancer therapy strategy

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.

Promise of spatially resolved omics for tumor research

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.

A temporo-spatial pharmacometabolomics method to characterize pharmacokinetics and pharmacodynamics in the brain microregions by using ambient mass spectrometry imaging

The brain is the most advanced organ with various complex structural and functional microregions. It is often challenging to understand what and where the molecular events would occur for a given drug treatment in the brain. Herein, a temporo-spatial pharmacometabolomics method was proposed based on ambient mass spectrometry imaging and was applied to evaluate the microregional effect of olanzapine(OLZ) on brain tissue and demonstrate its effectiveness in characterizing the microregional pharmacokinetics and pharmacodynamics of OLZ for improved understanding of the molecular mechanism of drugs acting on the microregions of the brain. It accurately and simultaneously illustrated the levels dynamics and microregional distribution of various substances, including exogenous drugs and its metabolites, as well as endogenous functional metabolites from complicated brain tissue. The targeted imaging analysis of the prototype drug and its metabolites presented the absorption, distribution, metabolism, and excretion characteristics of the drug itself. Moreover, the endogenous functional metabolites were identified along with the associated therapeutic and adverse effects of the drug, which can reflect the pharmacodynamics effect on the microregional brain. Therefore, this method is significant in elucidating and understanding the molecular mechanism of central nervous system drugs at the temporo and spatial metabolic level of system biology.

Rapid and sensitive liquid chromatography–tandem mass spectrometric method for the quantitative determination of potentially harmful substance 5,5′-oxydimethylenebis (2-furfural) in traditional Chinese medicine injections

With the rapid development and wide application of traditional Chinese medicine injection(TCMI), a number of adverse events of some TCMIs have incessantly been reported and have drawn broad attention in recent years. Establishing effective and practical analytical methods for safety evaluation and quality control of TCMI can help to improve the safety of TCMIs in clinical applications. In this study, a sensitive and rapid high-performance liquid chromatography–tandem mass spectrometry(HPLC–MS/MS)method has been developed and validated for the quantitative determination of potentially harmful substance5,5′-oxydimethylenebis(2-furfural, OMBF) in TCMI samples. Chromatographic separation was performed on a C18 reversed-phase column(150 mm × 2.1 mm, 5 μm) by gradient elution, using methanol–water containing 0.1% formic acid as mobile phase at the flow rate of 0.3 m L/min. MS/MS detection was performed on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction-monitoring mode. The method was sensitive with a limit of quantification of 0.3 ng/m L and linear over the range of 0.3–30 ng/m L(r = 0.9998). Intra-and inter-day precision for analyte was o9.52% RSD withrecoveries in the range 88.0–109.67% at three concentration levels. The validated method was successfully applied to quantitatively determine the compound OMBF in TCMIs and glucose injections. Our study indicates that this method is simple, sensitive, practicable and reliable, and could be applied for safety evaluation and quality control of TCMIs and glucose injections.

A rapid and sensitive UPLC–MS/MS method for quantitative determination of arformoterol in rat plasma, lung and trachea tissues

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry（UPLC–MS/MS） method was developed and fully validated for determination of arformoterol in rat plasma, lung and trachea tissues. The chromatographic separation was performed on an Agilent XDB C8 column with gradient elution by using acetonitrile and 0.1% formic acid water. The method presented high sensitivity（LLOQ of 1.83 pg/mL for plasma and 3.90 pg/mL for lung and trachea tissue homogenates） and great linearity over the range of 1.83–458 pg/mL for plasma, 13.9–1560 pg/mL for lung and trachea tissue homogenates. No carry over effect and matrix effect were observed. The intra-and inter-day precision/accuracy were within ±15% at three quality control concentration levels. This robust method was successfully applied to the pharmacokinetic, lung and trachea tissue distribution study after inhalation administration of arformoterol tartrate inhalation solution（50 m g/kg）. The in vivo information indicate that arformoterol can be rapidly absorbed into blood through respiratory systems, lung and trachea tissue maintain a certain amount of arformoterol in 1 h after dosing.

Spatial metabolomics highlights metabolic reprogramming in acute myeloid leukemia mice through creatine pathway

Acute myeloid leukemia(AML)is recognized as an aggressive cancer that is characterized by significant metabolic reprogramming.Here,we applied spatial metabolomics to achieve high-throughput,in situ identification of metabolites within the liver metastases of AML mice.Alterations at metabolite and protein levels were further mapped out and validated by integrating untargeted metabolomics and proteomics.This study showed a downregulation in arginine's contribution to polyamine biosynthesis and urea cycle,coupled with an upregulation of the creatine metabolism.The upregulation of creatine synthetases Gatm and Gamt,as well as the creatine transporter Slc6a8,resulted in a marked accumulation of creatine within tumor foci.This process further enhances oxidative phosphorylation and glycolysis of leukemia cells,thereby boosting ATP production to foster proliferation and infiltration.Importantly,we discovered that inhibiting Slc6a8 can counter these detrimental effects,offering a new strategy for treating AML by targeting metabolic pathways.

Plasma metabolomics combined with mass spectrometry imaging reveals crosstalk between tumor and plasma in gastric cancer genesis and metastasis

Gastric Carcinoma(GC)is a highly fatal malignant tumor with a poor prognosis.Its elevated mortality rates are primarily due to its proclivity for late-stage metastasis.Exploring the metabolic interactions between tumor microenvironment and the systemic bloodstream could help to clearly understand the mechanisms and identify precise biomarkers of tumor growth,proliferation,and metastasis.In this study,an integrative approach that combines plasma metabolomics with mass spectrometry imaging of tumor tissue was developed to investigate the global metabolic landscape of GC tumorigenesis and metastasis.The results showed that the oxidized glutathione to glutathione ratio(GSSH/GSH)became increased in non-distal metastatic GC(M0),which means an accumulation of oxidative stress in tumor tissues.Furthermore,it was found that the peroxidation of polyunsaturated fatty acids,such as 9,10-EpOMe,9-HOTrE,etc.,were accelerated in both plasma and tumor tissues of distal metastatic GC(M1).These changes were further confirmed the potential effect of CYP2E1 and GGT1 in metastatic potential of GC by mass spectrometry imaging(MSI)and immunohistochemistry(IHC).Collectively,our findings reveal the integrated multidimensional metabolomics approach is a clinical useful method to unravel the bloodtumor metabolic crosstalk,illuminate reprogrammed metabolic networks,and provide reliable circulating biomarkers.

Spatiotemporally resolved metabolomics and isotope tracing reveal CNS drug targets

Deconvolution of potential drug targets of the central nervous system(CNS)is particularly challenging because of the complicated structure and function of the brain.Here,a spatiotemporally resolved metabolomics and isotope tracing strategy was proposed and demonstrated to be powerful for deconvoluting and localizing potential targets of CNS drugs by using ambient mass spectrometry imaging.This strategy can map various substances including exogenous drugs,isotopically labeled metabolites,and various types of endogenous metabolites in the brain tissue sections to illustrate their microregional distribution pattern in the brain and locate drug action-related metabolic nodes and pathways.The strategy revealed that the sedative-hypnotic drug candidate YZG-331 was prominently distributed in the pineal gland and entered the thalamus and hypothalamus in relatively small amounts,and can increase glutamate decarboxylase activity to elevateγ-aminobutyric acid(GABA)levels in the hypothalamus,agonize organic cation transporter 3 to release extracellular histamine into peripheral circulation.These findings emphasize the promising capability of spatiotemporally resolved metabolomics and isotope tracing to help elucidate the multiple targets and the mechanisms of action of CNS drugs.

A high-performance bio-tissue imaging method using air flow-assisted desorption electrospray ionization coupled with a high-resolution mass spectrometer

Mass spectrometry imaging (MSI) technology can simultaneously obtain the spatial distribution of thousands of chemical compounds and has unique advantages compared to other techniques that allow mapping the surface of bio-tissue. Here, we combined an air flow-assisted desorption electrospray ionization (AFADESI) MSI device with a high-resolution mass spectrometer to optimize the system parameters and achieve more accurate spatial distribution characteristics for compounds of interest while investigating bio-tissue sections. The platform set-up, required instrumentation, sample pretreatment, parameter optimization and bio-tissue characterization are described and discussed.Finally, the parameter conditions that can provide optimal ionic intensity and enhanced resolution were confirmed. The reasonable resolution and sensitivity improvements of AFADESI-MSI have been achieved through tandem a high-resolution mass spectrometer system, therefore, it would be a promising technique for the bio-tissue imaging analysis.

Self-assembly of Pyrene-modified Rhomboidal Metalloden- drimers via Directional Metal-ligand Bonding Approach

The design and self-assembly of pyrene-modified rhomboidal metallodendrimers R1--R6 via directional metal-ligand bonding approach is described. By employing pyrene-containing 120° di-Pt（II） acceptor and appropri- ate 60° dendritic dipyridyl donors, a variety of [G-1]--[G-3] pyrene-modified rhomboidal metallodendrimers with well-defined shape and size were prepared under mild conditions in high yields. The supramolecular dendrimers were characterized with multinuclear NMR （1H and 31p） and mass spectrometry （CSI-TOF-MS）. lsotopically re- solved mass spectrometry data support the existence of the pyrene-modified dendrimers with rhomboidal cavities, and NMR data were consistent with the formation of all ensembles. The shape and size of all rhomboidal den- drimers were investigated with the PM6 semiempirical molecular orbital method. Their primary photochemical properties were studied as well.

Design and characterizing of robust probes for enhanced mass spectrometry imaging and spatially resolved metabolomics

As for the emerging and cut edge spatially resolved metabolomics,mass spectrometry imaging(MSI)is a powerful tool that can map thousands of metabolites from bio-tissue sections without chemical labels.However,the stability,sensitivity and spatial resolution of MSI are always limited by the performance of its ionization probe.Herein,two types of probes(fine probe(P-100)and large probe(P-200))were designed and characterized to perform air-flow assisted desorption electrospray ionization(AFA-DESI)MSI analysis for spatially resolved metabolomics.It was determined that the spray introduced by P-100 was homogenous and stable under the spray solvent at a flow rate of 5-10μL/min,while P-200 can endure a high flow rate of up to 10-30μL/min.Moreover,the MSI images were acquired by AFA-DESI-MSI with P-100 from rat brain tissue section and with P-200 from whole-body tissue section of mouse,and these results presented unambiguous tissue structure with the distribution information of numerous metabolites.Furthermore,the spatially resolved metabolomic analysis of tumor tissue was successfully realized to discover the tumor associated biomarkers.As the key parts of AFA-DESI-MSI system,it has been demonstrated that the designed probs have excellent performance for spatially resolved metabolomics,and it will further promote its application in life science,and drug research and development.