Matrix-assisted Laser Desorption/ionization Imaging Research Articles

Abstract 6188: Pathomics of prostate cancer incorporating histology and mass spectrometry imaging

Abstract Background: Recent evidence shows that interactions between tumor cells, immune cells, and extracellular matrix (ECM) proteins in the stroma play an important role in prostate cancer (PCa) progression. However, to date these factors are not accounted for in clinical evaluation of PCa patients. Our team has developed methods based on matrix-assisted laser desorption/ionization (MALDI) imaging to identify sets of ECM glycan and collagen molecules associated with PCa. In this study, we leverage computational imaging methods and pathomic textural features to assess the potential of MALDI imaging to guide the identification of regions in PCa hematoxylin and eosin stain (H&E) images as tumor or non-tumor. Methods: We used a cohort of PCa patients (n=10), who have undergone radical prostatectomy, with grade groups GG3−GG5, and with available H&E and N-glycan MALDI images. We used five MALDI images per patient representing the distribution of N-glycan species at 1419, 2539, 2686, 1663, and 1809 m/z. For each MALDI image, an N-glycan mask was generated and co-registered with the corresponding H&E image via computational imaging methods (i.e., Otsu thresholding, largest connected components labeling, sequential affine and B-spline registration using mutual information). To quantify the overlap of each mask with tumor regions, we calculated Dice scores between the masks and the pathology annotation in H&E. To assess differences in imaging patterns of regions indicated by the different masks, we extracted a set of pathomic textural features (n=70) from the H&E image within each mask using PyRadiomics, and we performed Wilcoxon signed rank tests across all pathomic textural features for all paired combinations of the five masks. Results: The mean Dice scores between the pathology tumor annotation and the N-glycan masks from 1419 (0.56 [0.44‒0.65]) and 2539 m/z (0.64 [0.59‒0.82]) was significantly higher (p<0.008) than the Dice score of 1663 (0.24 [0.17‒0.36]), 1809 (0.31 [0.13‒0.45]), and 2686 m/z (0.32 [0.19‒0.37]). This suggests a large overlap between 1419 and 2539 m/z and tumor, and large overlap between 1663 and 1809 m/z and non-tumor regions. Consistent with these results, our pathomic textural analysis showed that 50 out of 70 features were significantly different (p<0.05) between the masks derived from 1419 and 1663 m/z, 52 between 1419 and 1809 m/z, 62 between 2539 and 1663 m/z, and 57 between 2539 and 1809 m/z (p<0.05). Moreover, there were only 23 significantly different features between 1419 and 2539 m/z (p<0.05), and 0 features between 1663 and 1809 m/z. Further evaluation is needed for 2686 m/z for which a mean of 54 [45‒64] significantly different features (p<0.05) from all other masks were found. Conclusions: We provide preliminary evidence that MALDI imaging can elucidate tissue properties and guide pathomic textural analysis in H&E images, with potential applications in treatment guidance and prognostication of PCa. Citation Format: José Marcio Luna, Hani Nakhoul, Cody Weimholt, Eric H. Kim, Sheng-Kwei Song, Peggi M. Angel, Richard R. Drake, Joseph E. Ippolito. Pathomics of prostate cancer incorporating histology and mass spectrometry imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6188.

Formation of multiple ion types during MALDI imaging mass spectrometry analysis of Mitragyna speciosa alkaloids in dosed rat brain tissue

Mitragyna speciosa, more commonly known as kratom, has emerged as an alternative to treat chronic pain and addiction. However, the alkaloid components of kratom, which are the major contributors to kratom's pharmaceutical properties, have not yet been fully investigated. In this study, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to map the biodistribution of three alkaloids (corynantheidine, mitragynine, and speciogynine) in rat brain tissues. The alkaloids produced three main ion types during MALDI analysis: [M + H]+, [M − H]+, and [M − 3H]+. Contrary to previous reports suggesting that the [M − H]+ and [M − 3H]+ ion types form during laser ablation, these ion types can also be produced during the MALDI matrix application process. Several strategies are proposed to accurately map the biodistribution of the alkaloids. Due to differences in the relative abundances of the ions in different biological regions of the tissue, differences in ionization efficiencies of the ions, and potential overlap of the [M − H]+ and [M − 3H]+ ion types with endogenous metabolites of the same empirical formula, a matrix that mainly produces the [M + H]+ ion type is optimal for accurate mapping of the alkaloids. Alternatively, the most abundant ion type can be mapped or the intensities of all ion types can be summed together to generate a composite image. The accuracy of each of these approaches is explored and validated.

Nitro Indole Derivatives as Novel Dual-Polarity Matrices for MALDI Mass Spectrometry and Imaging with Broad Applications.

A new matrix framework is presented in this study for the improved ionization efficiency of complex mixtures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry/imaging. Five nitro indole (NI) derivatives [3-methyl-4-nitro-1H-indole (3,4-MNI), 3-methyl-6-nitro-1H-indole (3,6-MNI), 2,3-dimethyl-4-nitro-1H-indole (2,3,4-DMNI), 2,3-dimethyl-6-nitro-1H-indole (2,3,6-DMNI), and 4-nitro-1H-indole (4-NI)] were synthesized and shown to produce both positive and negative ions with a broad class of analytes as MALDI matrices. NI matrices were compared to several common matrices, such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxylcinnamic acid (CHCA), sinapinic acid (SA), 1,5-diaminonaphthelene (1,5-DAN), and 9-aminoacridine (9-AA), for the analysis of lipid, peptide, protein, glycan, and perfluorooctanesulfonic acid (PFOS) compounds. 3,4-MNI demonstrated the best performance among the NI matrices. This matrix resulted in reduced ion suppression and better detection sensitivity for complex mixtures, for example, egg lipids/milk proteins/PFOS in tap water, while 2,3,6-DMNI was the best matrix for blueberry tissue imaging. Several important aspects of this work are reported: (1) dual-polarity ion production with NI matrices and complex mixtures; (2) quantitative analysis of PFOS with a LOQ of 0.5 ppb in tap water and 0.05 ppb in MQ water (without solid phase extraction enrichment), with accuracy and precision within 5%; (3) MALDI imaging with 2,3,6-DMNI as a matrix for plant metabolite/lipid identification with ionization enhancement in the negative ion mode m/z 600-900 region; and (4) development of a thin film deposition under/above tissue method for MALDI imaging with a vacuum sublimation matrix on a high-vacuum MALDI instrument.

Spatial Omics Reveals that Cancer-Associated Glycan Changes Occur Early in Liver Disease Development in a Western Diet Mouse Model of MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disease and comprises different stages of liver damage; it is significantly associated with obese and overweight patients. Untreated MASLD can progress to life-threatening end-stage conditions, such as cirrhosis and liver cancer. N-Linked glycosylation is one of the most common post-translational modifications in the cell surface and secreted proteins. N-Linked glycan alterations have been established to be signatures of liver diseases. However, the N-linked glycan changes during the progression of MASLD to liver cancer are still unknown. Here, we induced different stages of MASLD in mice and liver-cancer-related phenotypes and elucidated the N-glycome profile during the progression of MASLD by quantitative and qualitative profiling in situ using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Importantly, we identified specific N-glycan structures including fucosylated and highly branched N-linked glycans at very early stages of liver injury (steatosis), which in humans are associated with cancer development, establishing the importance of these modifications with disease progression. Finally, we report that N-linked glycan alterations can be observed in our models by MALDI-IMS before liver injury is identified by histological analysis. Overall, we propose these findings as promising biomarkers for the early diagnosis of liver injury in MASLD.

Review on MALDI Imaging for Direct Tissue Imaging and its Application in Pharmaceutical Research

Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging has emerged as a powerful analytical technique, enabling direct tissue imaging in pharmaceutical research. This review provides an in-depth exploration of the principles, methodologies, and applications of MALDI imaging in the context of studying drug distribution and molecular changes within tissues. provides a structured approach to exploring the principles, methodologies, applications, challenges, and future directions of MALDI Imaging in pharmaceutical research. Each section aims to contribute to a comprehensive understanding of the technique’s significance and potential for transformative contributions to the field. The study begins by elucidating the underlying principles of MALDI imaging, highlighting the role of matrix-assisted laser desorption/ionization in generating spatially resolved mass spectra from biological samples. Special emphasis is placed on advancements in instrumentation and sample preparation techniques that have enhanced the spatial resolution and sensitivity of MALDI imaging. The application of MALDI imaging in pharmaceutical research is comprehensively explored, focusing on its pivotal role in drug development, pharmacokinetic studies, and toxicity assessments. Case studies and examples illustrate how MALDI imaging facilitates the visualization of drug distribution within tissues, offering valuable insights into drug metabolism, biodistribution, and pharmacodynamics.

FluoMALDI Microscopy: Matrix Co-Crystallization Simultaneously Enhances Fluorescence and MALDI Imaging.

Here, the authors report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. The approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm simplifies registration by avoiding physical changes between fluorescence and MALDI imaging, allowing to image the exact same cells in tissues with both modalities. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. The authorsdemonstrate feasibility of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.

A Guide to MALDI Imaging Mass Spectrometry for Tissues.

Imaging mass spectrometry is a well-established technology that can easily and succinctly communicate the spatial localization of molecules within samples. This review communicates the recent advances in the field, with a specific focus on matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) applied on tissues. The general sample preparation strategies for different analyte classes are explored, including special considerations for sample types (fresh frozen or formalin-fixed,) strategies for various analytes (lipids, metabolites, proteins, peptides, and glycans) and how multimodal imaging strategies can leverage the strengths of each approach is mentioned. This work explores appropriate experimental design approaches and standardization of processes needed for successful studies, as well as the various data analysis platforms available to analyze data and their strengths. The review concludes with applications of imaging mass spectrometry in various fields, with a focus on medical research, and some examples from plant biology and microbe metabolism are mentioned, to illustrate the breadth and depth of MALDI IMS.

A Comprehensive Comparison of Tissue Processing Methods for High-Quality MALDI Imaging of Lipids in Reconstructed Human Epidermis.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has become an important tool for skin analysis, as it allows the simultaneous detection and localization of diverse molecular species within a sample. The use of in vivo and ex vivo human skin models is costly and presents ethical issues; therefore, reconstructed human epidermis (RHE) models, which mimic the upper part of native human skin, represent a suitable alternative to investigate adverse effects of chemicals applied to the skin. However, there are few publications investigating the feasibility of using MALDI MSI on RHE models. Therefore, the aim of this study was to investigate the effect of sample preparation techniques, i.e., substrate, sample thickness, washing, and matrix recrystallization, on the quality of MALDI MSI for lipids analysis of the SkinEthic RHE model. Images were generated using an atmospheric pressure MALDI source coupled to a high-resolution mass spectrometer with a pixel size of 5 μm. Masses detected in a defined region of interest were analyzed and annotated using the LipostarMSI platform. The results indicated that the combination of (1) coated metallic substrates, such as APTES-coated stainless-steel plates, (2) tissue sections of 6 μm thickness, and (3) aqueous washing before HCCA matrix spraying (without recrystallization), resulted in images with a significant signal intensity as well as numerous m/z values. This refined methodology using AP-MALDI coupled to a high-resolution mass spectrometer should improve the current sample preparation workflow to evaluate changes in skin composition after application of dermatocosmetics.

Imaging and Structural Characterization of Phosphatidylcholine Isomers from Rat Brain Tissue Using Sequential Collision-Induced Dissociation/Electron-Induced Dissociation.

The chemical complexity of biological tissues creates challenges in the analysis of lipids via imaging mass spectrometry. The presence of isobaric and isomeric compounds introduces chemical noise that makes it difficult to unambiguously identify and accurately map the spatial distributions of these compounds. Electron-induced dissociation (EID) has previously been shown to profile phosphatidylcholine (PCs) sn-isomers directly from rat brain tissue in matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry. However, the acquisition of true pixel-by-pixel images, as opposed to regional profiling measurements, using EID is difficult due to low fragmentation efficiency and precursor ion signal dilution into multiple fragment ion channels, resulting in low sensitivity. In this work, we have developed a sequential collision-induced dissociation (CID)/EID method to visualize the distribution of sn-isomers in MALDI imaging mass spectrometry experiments. Briefly, CID is performed on sodium-adducted PCs, which results in facile loss of the phosphocholine headgroup. This ion is then subjected to an EID analysis. Since the lipid headgroup is removed prior to EID, a major fragmentation pathway common to EID ion activation is eliminated, resulting in a more sensitive analysis. This sequential CID/EID workflow generates sn-specific fragment ions allowing for the assignment of the sn-positions. Carbon-carbon double-bond (C═C) positions are also localized along the fatty acyl tails by the presence of a 2 Da shift pattern in the fragment ions arising from carbon-carbon bond cleavages. Moreover, the integration of the CID/EID method into MALDI imaging mass spectrometry enables the mapping of the absolute and relative distribution of sn-isomers at every pixel. The localized relative abundances of sn-isomers vary throughout brain substructures and likely reflect different biological functions and metabolism.

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Validating MALDI-IMS Feasibility in Ex Vivo Brain Slices.

Matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) generates unique mass spectra in X/Y coordinates across a tissue sample, thus allowing for the spatial detection and relative quantification of biologic compounds in situ. The soft ionization of MALDI-IMS makes it an ideal technique for high-resolution imaging of complex lipid species. Lipid-based spatial chemical maps derived from MALDI-IMS provide critical insight into the unique molecular profiles of a variety of neurologic diseases. Ex vivo brain slice preparations are a prominent alternative to in vivo animal models for studying many different neurologic conditions. For the first time, we present a feasible protocol for achieving reproducible lipidomic MALDI-IMS data from ex vivo rat brain slices and provide evidence that ex vivo brain slices maintain spatiochemical lipidomic profiles representative of an intact whole brain. We conducted a methods comparison assessing the lipid profiles within the neocortex, striatum, and corpus callosum between coronal sections taken from ex vivo brain slices and the current gold standard tissue preparation method, fresh frozen whole brains. For the first time we demonstrate a technique by which 400 μm ex vivo brain slices can be extracted from an imaging chamber and prepared for MALDI-IMS in a way that preserves their lipidomic integrity. We demonstrate the feasibility of MALDI-IMS in ex vivo brain slices and provide a roadmap for MALDI-IMS utilization in uncharted neuroscience fields.

Shotgun proteomics identification of proteins expressed in the Descemet’s membrane of patients with Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet’s membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεβ) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

Single-filament imaging mass spectrometry lipidomics in Arthrospira platensis.

Elucidating intra-organismal biochemical and lipid organization in photosynthetic biological cell factories of filamentous cyanobacteria, such as Arthrospira platensis (Spirulina), is important for tracking physiological response mechanisms during growth. Little is known about the filaments' biochemical organization and cellular structure and no label-free imaging techniques exist that provide molecular mapping. We applied ultrahigh-resolution mass spectrometry (MS) with matrix-assisted laser desorption ionization (MALDI) imaging to immobilized Spirulina filaments to investigate the localization of lipids across distinct physiological regions. We optimized matrix selection and deposition methods with the goal of facilitating high spatial, and intra-filament, resolution using untargeted multivariate statistical spectral deconvolution across MS pixels. Our results demonstrate an improved two-step matrix application with an optimized procedure for intra-organismal lipid profiling to improve analyte sensitivity and achieve higher spatial resolution. We evaluate several conventional matrices, namely 2,5-dihydroxybenzoic acid (DHB), superDHB (sDHB), 1,5-diaminonaphthalene (DAN), and a 50:50 mix of DHB and sDHB, and compare delineation and pixel-based elucidation of intra-filament lipidomics. We identified a total of 1626 features that could be putatively assigned a lipid-like formula based on database query and 46 unique features, with associated lipid assignments that were significantly distinct in their intra-filament location. MALDI imaging MS with untargeted statistical spectral deconvolution was used to visualize intra-filament lipidomics organization in Spirulina filaments. Improvements in matrix deposition, including sequential sublimation and pneumatic spraying, increased signal abundance at high spatial resolution and allowed for identification of distinct lipid composition regions. This work outlines a methodology that may be used for micro-ecological untargeted molecular phenotyping.

MALDImID: Spatialomics R package and Shiny app for more specific identification of MALDI imaging proteolytic peaks using LC-MS/MS-based proteomic biomarker discovery data.

Matrix-assisted laser desorption/ionization (MALDI) imaging of proteolytic peptides from formalin-fixed paraffin embedded (FFPE) tissue sections could be integrated in the portfolio of molecular pathologists for protein localization and tissue classification. However, protein identification can be very tedious using MALDI-time-of-flight (TOF) and post-source decay (PSD)-based fragmentation. Hereby, we implemented an R package and Shiny app to exploit liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic biomarker discovery data for more specific identification of peaks observed in bottom-up MALDI imaging data. The package is made available under the GPL 3 license. The Shiny app can directly be used at the following address: https://biosciences.shinyapps.io/Maldimid.

MALDI Imaging, a Powerful Multiplex Approach to Decipher Intratumoral Heterogeneity: Combined Hepato-Cholangiocarcinomas as Proof of Concept

Combined hepato-cholangiocarcinomas (cHCC-CCA) belong to the spectrum of primary liver carcinomas, which include hepatocellular carcinomas (HCC) and intrahepatic cholangiocarcinomas (iCCA) at both ends of the spectrum. Mainly due to the high intratumor heterogeneity of cHCC-CCA, its diagnosis and pathological description remain challenging. Taking advantage of in situ non-targeted molecular mapping provided by MALDI (Matrix Assisted Laser Desorption Ionization) imaging, we sought to develop a multiscale and multiparametric morphological approach, integrating molecular and conventional pathological analysis. MALDI imaging was applied to five representative cases of resected cHCC-CCA. Principal component analysis and segmentations with MALDI imaging techniques identified areas related to either iCCA or HCC and also hidden tumor areas not visible microscopically. In addition, the overlap between MALDI segmentation and immunostaining provided a comprehensive description of cHCC-CCA tumor heterogeneity by identifying transitional and micro-metastatic areas. Moreover, a list of peptides derived from in silico digestion was obtained for each immunohistochemical marker and was matched within the peptide peak list acquired by MALDI. Comparison of immunostaining images with ions from in silico digestion revealed an accurate identification of iCCA and HCC areas. Our study provides further evidence on the performance of MALDI imaging in exploring intratumor heterogeneity and offering virtual multiplex immunostaining through a single acquisition.

A Multimodal SIMS/MALDI Mass Spectrometry Imaging Source with Secondary Electron Imaging Capabilities for Use with timsTOF Instruments.

Mass spectrometry imaging (MSI) is a surface analysis technique that produces chemical images and is commonly used for biological and biomedical research. Multimodal imaging combines multiple imaging modes in order to get a more comprehensive view of a sample. Multimodal MSI images are often acquired using multiple MSI instruments, which leads to issues regarding image registration and increases the chance of sample damage or degradation during sample transfer. These problems can be solved by using a single instrument that can image in multiple modes. In order to improve the efficiency of multimodal imaging and investigate complementary modes of MSI, we have modified a prototype Bruker timsTOF fleX by adding secondary ion mass spectrometry (SIMS) and secondary electron (SE) imaging capabilities while preserving the ability to perform matrix-assisted laser desorption/ionization (MALDI). We show multimodal images collected on this instrument that required only trivial registration and were acquired without sample transfer between imaging trials. Furthermore, we characterize the performance of SIMS, SE, and MALDI imaging and compare the performance of the modified instrument to a commercial timsTOF fleX.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2019-2020.

This review is the tenth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2020. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. The review is basically divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of arrays. (2) Applications to various structural types such as oligo- and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other areas such as medicine, industrial processes and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. The reported work shows increasing use of incorporation of new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented nearly 40 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show little sign of diminishing.

Noncanonical atherosclerosis as the driving force in tricuspid aortic valve associated aneurysms - A trace collection

Pathogenic mechanisms in degenerative thoracic aortic aneurysms (TAA) are still unclear. There is an ongoing debate about whether TAAs are caused by uniform or distinct processes, which would obviously have a major impact on future treatment strategies. Clearly, the ultimate outcome of TAA subgroups associated with a tricuspid aortic valve (TAV) or a bicuspid aortic valve (BAV) is the same, namely a TAA. Based on results from our own and others' studies, we decided to compare the different TAAs (TAV and BAV) and controls using a broad array of analyses, i.e., metabolomic analyses, gene expression profiling, protein expression analyses, histological characterization, and matrix-assisted laser desorption ionization imaging. Central findings of the present study are the presence of noncanonical atherosclerosis, pathological accumulation of macrophages, and disturbances of lipid metabolism in the aortic media. Moreover, we have also found that lipid metabolism is impaired systemically. Importantly, all of the above-described phenotypes are characteristic for TAV-TAA only, and not for BAV-TAA. In summary, our results suggest different modes of pathogenesis in TAV- and BAV-associated aneurysms. Intimal atherosclerotic changes play a more central role in TAV-TAA formation than previously thought, particularly as the observed alterations do not follow classical patterns. Atherosclerotic alterations are not limited to the intima but also affect and alter the TAV-TAA media. Further studies are needed to i) clarify patho-relevant intima-media interconnections, ii) define the origin of the systemic alteration of lipid metabolism, and iii) to define valid biomarkers for early diagnosis, disease progression, and successful treatments in TAV-TAAs.

Spatially Resolved Molecular Approaches for the Characterisation of Non-Invasive Follicular Tumours with Papillary-like Features (NIFTPs).

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)-Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI-MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS-MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

MALDI Imaging Mass Spectrometry of High-Grade Gliomas: A Review of Recent Progress and Future Perspective.

Glioblastoma (GBM) is the most common malignancy of the brain with a relatively short median survival and high mortality. Advanced age, high socioeconomic status, exposure to ionizing radiation, and other factors have been correlated with an increased incidence of GBM, while female sex hormones, history of allergies, and frequent use of specific drugs might exert protective effects against this disease. However, none of these explain the pathogenesis of GBM. The most recent WHO classification of CNS tumors classifies neoplasms based on their histopathological and molecular characteristics. Modern laboratory techniques, such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, enable the comprehensive metabolic analysis of the tissue sample. MALDI imaging is able to characterize the spatial distribution of a wide array of biomolecules in a sample, in combination with histological features, without sacrificing the tissue integrity. In this review, we first provide an overview of GBM epidemiology, risk, and protective factors, as well as the recent WHO classification of CNS tumors. We then provide an overview of mass spectrometry workflow, with a focus on MALDI imaging, and recent advances in cancer research. Finally, we conclude the review with studies of GBM that utilized MALDI imaging and offer our perspective on future research.