Advances in mass spectrometry imaging for plant metabolomics-Expanding the analytical toolbox.

Mass spectrometry imaging (MSI) has become increasingly popular in plant science due to its ability to characterize complex chemical, spatial, and temporal aspects of plant metabolism. Over the past decade, as the emerging and unique features of various MSI techniques have continued to support new discoveries in studies of plant metabolism closely associated with various aspects of plant function and physiology, spatial metabolomics based on MSI techniques has positioned it at the forefront of plant metabolic studies, providing the opportunity for far higher resolution than was previously available. Despite these efforts, profound challenges at the levels of spatial resolution, sensitivity, quantitative ability, chemical confidence, isomer discrimination, and spatial multi-omics integration, undoubtedly remain. In this Perspective, we provide a contemporary overview of the emergent MSI techniques widely used in the plant sciences, with particular emphasis on recent advances in methodological breakthroughs. Having established the detailed context of MSI, we outline both the golden opportunities and key challenges currently facing plant metabolomics, presenting our vision as to how the enormous potential of MSI technologies will contribute to progress in plant science in the coming years.

MSIpixel: a fully automated pipeline for compound annotation and quantitation in mass spectrometry imaging experiments.

Mass spectrometry imaging (MSI) is commonly used to map the spatial distribution of small molecules within complex biological matrices. One of the major challenges in imaging MS-based spatial metabolomics is molecular identification and metabolite annotation, to address this limitation, annotation is often complemented with parallel bulk LC-MS2-based metabolomics to confirm and validate identifications. Here we applied MSI method, utilizing data-dependent acquisition, to visualize and identify unknown molecules in a single instrument run. To reach this aim we developed MSIpixel, a fully automated pipeline for compound annotation and quantitation in MSI experiments. It overcomes challenges in molecular identification, and improving reliability and comprehensiveness in MSI-based spatial metabolomics.

Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances.

With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.

Modern machine-learning applications in ambient ionization mass spectrometry.

This article provides a comprehensive overview of the applications of methods of machine learning (ML) and artificial intelligence (AI) in ambient ionization mass spectrometry (AIMS). AIMS has emerged as a powerful analytical tool in recent years, allowing for rapid and sensitive analysis of various samples without the need for extensive sample preparation. The integration of ML/AI algorithms with AIMS has further expanded its capabilities, enabling enhanced data analysis. This review discusses ML/AI algorithms applicable to the AIMS data and highlights the key advancements and potential benefits of utilizing ML/AI in the field of mass spectrometry, with a focus on the AIMS community.

Metabolic insights from mass spectrometry imaging of biofilms: A perspective from model microorganisms.

Biofilms are dense aggregates of bacterial colonies embedded inside a self-produced polymeric matrix. Biofilms have received increasing attention in medical, industrial, and environmental settings due to their enhanced survival. Their characterization using microscopy techniques has revealed the presence of structural and cellular heterogeneity in many bacterial systems. However, these techniques provide limited chemical detail and lack information about the molecules important for bacterial communication and virulence. Mass spectrometry imaging (MSI) bridges the gap by generating spatial chemical information with unmatched chemical detail, making it an irreplaceable analytical platform in the multi-modal imaging of biofilms. In the last two decades, over 30 species of biofilm-forming bacteria have been studied using MSI in different environments. The literature conveys both analytical advancements and an improved understanding of the effects of environmental variables such as host surface characteristics, antibiotics, and other species of microorganisms on biofilms. This review summarizes the insights from frequently studied model microorganisms. We share a detailed list of organism-wide metabolites, commonly observed mass spectral adducts, culture conditions, strains of bacteria, substrate, broad problem definition, and details of the MS instrumentation, such as ionization sources and matrix, to facilitate future studies. We also compared the spatial characteristics of the secretome under different study designs to highlight changes because of various environmental influences. In addition, we highlight the current limitations of MSI in relation to biofilm characterization to enable cross-comparison between experiments. Overall, MSI has emerged to become an important approach for the spatial/chemical characterization of bacterial biofilms and its use will continue to grow as MSI becomes more accessible.

Advancing rare cancer research by MALDI mass spectrometry imaging: Applications, challenges, and future perspectives in sarcoma.

MALDI mass spectrometry imaging (MALDI imaging) uniquely advances cancer research, by measuring spatial distribution of endogenous and exogenous molecules directly from tissue sections. These molecular maps provide valuable insights into basic and translational cancer research, including tumor biology, tumor microenvironment, biomarker identification, drug treatment, and patient stratification. Despite its advantages, MALDI imaging is underutilized in studying rare cancers. Sarcomas, a group of malignant mesenchymal tumors, pose unique challenges in medical research due to their complex heterogeneity and low incidence, resulting in understudied subtypes with suboptimal management and outcomes. In this review, we explore the applicability of MALDI imaging in sarcoma research, showcasing its value in understanding this highly heterogeneous and challenging rare cancer. We summarize all MALDI imaging studies in sarcoma to date, highlight their impact on key research fields, including molecular signatures, cancer heterogeneity, and drug studies. We address specific challenges encountered when employing MALDI imaging for sarcomas, and propose solutions, such as using formalin-fixed paraffin-embedded tissues, and multiplexed experiments, and considerations for multi-site studies and digital data sharing practices. Through this review, we aim to spark collaboration between MALDI imaging researchers and clinical colleagues, to deploy the unique capabilities of MALDI imaging in the context of sarcoma.

Spatial lipidomics of coronary atherosclerotic plaque development in a familial hypercholesterolemia swine model.

Coronary atherosclerosis is caused by plaque build-up, with lipids playing a pivotal role in its progression. However, lipid composition and distribution within coronary atherosclerosis remain unknown. This study aims to characterize lipids and investigate differences in lipid composition across disease stages to aid in the understanding of disease progression. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize lipid distributions in coronary artery sections (n = 17) from hypercholesterolemic swine. We performed histology on consecutive sections to classify the artery segments and to investigate colocalization between lipids and histological regions of interest in advanced plaque, including necrotic core and inflammatory cells. Segments were classified as healthy (n = 6), mild (n = 6), and advanced disease (n = 5) artery segments. Multivariate data analysis was employed to find differences in lipid composition between the segment types, and the lipids' spatial distribution was investigated using non-negative matrix factorization (NMF). Through this process, MALDI-MSI detected 473 lipid-related features. NMF clustering described three components in positive ionization mode: triacylglycerides (TAG), phosphatidylcholines (PC), and cholesterol species. In negative ionization mode, two components were identified: one driven by phosphatidylinositol(PI)(38:4), and one driven by ceramide-phosphoethanolamine(36:1). Multivariate data analysis showed the association between advanced disease and specific lipid signatures like PC(O-40:5) and cholesterylester(CE)(18:2). Ether-linked phospholipids and LysoPC species were found to colocalize with necrotic core, and mostly CE, ceramide, and PI species colocalized with inflammatory cells. This study, therefore, uncovers distinct lipid signatures correlated with plaque development and their colocalization with necrotic core and inflammatory cells, enhancing our understanding of coronary atherosclerosis progression.

eLIMS: Ensemble Learning-Based Spatial Segmentation of Mass Spectrometry Imaging to Explore Metabolic Heterogeneity.

Spatial segmentation is an essential processing method for image analysis aiming to identify the characteristic suborgans or microregions from mass spectrometry imaging (MSI) data, which is critical for understanding the spatial heterogeneity of biological information and function and the underlying molecular signatures. Due to the intrinsic characteristics of MSI data including spectral nonlinearity, high-dimensionality, and large data size, the common segmentation methods lack the capability for capturing the accurate microregions associated with biological functions. Here we proposed an ensemble learning-based spatial segmentation strategy, named eLIMS, that combines a randomized unified manifold approximation and projection (r-UMAP) dimensionality reduction module for extracting significant features and an ensemble pixel clustering module for aggregating the clustering maps from r-UMAP. Three MSI datasets are used to evaluate the performance of eLIMS, including mouse fetus, human adenocarcinoma, and mouse brain. Experimental results demonstrate that the proposed method has potential in partitioning the heterogeneous tissues into several subregions associated with anatomical structure, i.e., the suborgans of the brain region in mouse fetus data are identified as dorsal pallium, midbrain, and brainstem. Furthermore, it effectively discovers critical microregions related to physiological and pathological variations offering new insight into metabolic heterogeneity.

The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

Lipid imaging of Alzheimer's disease pathology.

Alzheimer's disease (AD) affects one in eight individuals over 65 and poses an immense societal challenge. AD pathology is characterized by the formation of beta-amyloid plaques and Tau tangles in the brain. While some disease-modifying treatments targeting beta-amyloid are emerging, the exact chain of events underlying the pathogenesis of this disease remains unclear. Brain lipids have long been implicated in AD pathology, though their role in AD pathogenesis remains not fully resolved. Significant advancements in mass spectrometry imaging (MSI) allow to detail spatial lipid regulations in biological tissues at the low um scale. In this issue, Huang et al. resolve spatial lipid patterns in human AD brain and genetic mouse models using desorption electrospray ionization (DESI)-based MSI integrated with other spatial techniques such as imaging mass cytometry of correlative protein signatures. Those spatial multiomics experiments identify plaque-associated lipid regulations that are dependent on progressing plaque pathology in both mouse models and the human brain. Of those lipid species, particularly pro-inflammatory lysophospholipids have been implicated in AD pathology through their interaction with both aggregating Aß and microglial activation through lipid sensing surface receptors. Together, this study provides further insight into how brain lipid homeostasis is linked to progressing AD pathology, and thereby highlights the potential of MSI-based spatial lipidomics as an emerging spatial biology technology for biomedical research.

Spatial omics reveals molecular changes in focal cortical dysplasia type II.

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.

Spatio-Temporal Study of Galactolipid Biosynthesis in Duckweed Using Mass Spectrometry Imaging and in vivo Isotope Labeling.

Isotope labeling coupled with mass spectrometry imaging (MSI) presents a potent strategy for elucidating the dynamics of metabolism at cellular resolution, yet its application to plant systems is scarce. It has the potential to reveal the spatio-temporal dynamics of lipid biosynthesis during plant development. In this study, we explore its application to galactolipid biosynthesis of an aquatic plant, Lemna minor, with D2O labeling. Specifically, matrix-assisted laser desorption/ionization-MSI data of two major galactolipids in L. minor, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, were studied after growing in 50% D2O media over a 15-day time period. When they were partially labeled after 5 d, three distinct binomial isotopologue distributions were observed corresponding to the labeling of partial structural moieties: galactose only, galactose and a fatty acyl chain and the entire molecule. The temporal change in the relative abundance of these distributions follows the expected linear pathway of galactolipid biosynthesis. Notably, their mass spectrometry images revealed the localization of each isotopologue group to the old parent frond, the intermediate tissues and the newly grown daughter fronds. Besides, two additional labeling experiments, (i) 13CO2 labeling and (ii) backward labeling of completely 50% D2O-labeled L. minor in H2O media, confirm the observations in forward labeling. Furthermore, these experiments unveiled hidden isotopologue distributions indicative of membrane lipid restructuring. This study suggests the potential of isotope labeling using MSI to provide spatio-temporal details in lipid biosynthesis in plant development.

Deep learning-based pseudo-mass spectrometry imaging analysis for precision medicine.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics provides systematic profiling of metabolic. Yet, its applications in precision medicine (disease diagnosis) have been limited by several challenges, including metabolite identification, information loss and low reproducibility. Here, we present the deep-learning-based Pseudo-Mass Spectrometry Imaging (deepPseudoMSI) project (https://www.deeppseudomsi.org/), which converts LC-MS raw data to pseudo-MS images and then processes them by deep learning for precision medicine, such as disease diagnosis. Extensive tests based on real data demonstrated the superiority of deepPseudoMSI over traditional approaches and the capacity of our method to achieve an accurate individualized diagnosis. Our framework lays the foundation for future metabolic-based precision medicine.

Co-existence of two antibiotic-producing marine bacteria: Pseudoalteromonas piscicida reduce gene expression and production of the antibacterial compound, tropodithietic acid, in Phaeobacter sp.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.

Next-generation pathology practices with mass spectrometry imaging.

Mass spectrometry imaging (MSI) is a powerful technique that reveals the spatial distribution of various molecules in biological samples, and it is widely used in pathology-related research. In this review, we summarize common MSI techniques, including matrix-assisted laser desorption/ionization and desorption electrospray ionization MSI, and their applications in pathological research, including disease diagnosis, microbiology, and drug discovery. We also describe the improvements of MSI, focusing on the accumulation of imaging data sets, expansion of chemical coverage, and identification of biological significant molecules, that have prompted the evolution of MSI to meet the requirements of pathology practices. Overall, this review details the applications and improvements of MSI techniques, demonstrating the potential of integrating MSI techniques into next-generation pathology practices.

What are we imaging? Software tools and experimental strategies for annotation and identification of small molecules in mass spectrometry imaging.

Mass spectrometry imaging (MSI) has become a widespread analytical technique to perform nonlabeled spatial molecular identification. The Achilles' heel of MSI is the annotation and identification of molecular species due to intrinsic limitations of the technique (lack of chromatographic separation and the difficulty to apply tandem MS). Successful strategies to perform annotation and identification combine extra analytical steps, like using orthogonal analytical techniques to identify compounds; with algorithms that integrate the spectral and spatial information. In this review, we discuss different experimental strategies and bioinformatics tools to annotate and identify compounds in MSI experiments. We target strategies and tools for small molecule applications, such as lipidomics and metabolomics. First, we explain how sample preparation and the acquisition process influences annotation and identification, from sample preservation to the use of orthogonal techniques. Then, we review twelve software tools for annotation and identification in MSI. Finally, we offer perspectives on two current needs of the MSI community: the adaptation of guidelines for communicating confidence levels in identifications; and the creation of a standard format to store and exchange annotations and identifications in MSI.

Advances in mass spectrometry imaging for toxicological analysis and safety evaluation of pharmaceuticals.

Safety issues caused by pharmaceuticals have frequently occurred worldwide, posing a tremendous threat to human health. As an essential part of drug development, the toxicological analysis and safety evaluation is of great significance. In addition, the risk of pharmaceuticals accumulation in the environment and the monitoring of the toxicity from natural medicines have also received ongoing concerns. Due to a lack of spatial distribution information provided by common analytical methods, analyses that provide spatial dimensions could serve as complementary safety evaluation methods for better prediction and evaluation of drug toxicity. With advances in technical solutions and software algorithms, mass spectrometry imaging (MSI) has received increasing attention as a popular analytical tool that enables the simultaneous implementation of qualitative, quantitative, and localization without complex sample pretreatment and labeling steps. In recent years, MSI has become more attractive, powerful, and sensitive and has been applied in several scientific fields that can meet the safety assessment requirements. This review aims to cover a detailed summary of the various MSI technologies utilized in the biomedical and pharmaceutical area, including technical principles, advantages, current status, and future trends. Representative applications and developments in the safety-related issues of different pharmaceuticals and natural medicines are also described to provide a reference for pharmaceutical research, improve rational clinical medicine use, and ensure public safety.

Neurotrauma investigation through spatial omics guided by mass spectrometry imaging: Target identification and clinical applications.

Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as military-related practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.

Desorption electrospray ionization mass spectrometry imaging in discovery and development of novel therapies.

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is one of the least specimen destructive ambient ionization mass spectrometry tissue imaging methods. It enables rapid simultaneous mapping, measurement, and identification of hundreds of molecules from an unmodified tissue sample. Over the years, since its first introduction as an imaging technique in 2005, DESI-MSI has been extensively developed as a tool for separating tissue regions of various histopathologic classes for diagnostic applications. Recently, DESI-MSI has also emerged as a versatile technique that enables drug discovery and can guide the efficient development of drug delivery systems. For example, it has been increasingly employed for uncovering unique patterns of in vivo drug distribution, the discovery of potentially treatable biochemical pathways, revealing novel druggable targets, predicting therapeutic sensitivity of diseased tissues, and identifying early tissue response to pharmacological treatment. These and other recent advances in implementing DESI-MSI as the tool for the development of novel therapies are highlighted in this review.