Laser Desorption Ionization-mass Spectrometry Imaging Research Articles

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Multimodal Spectroscopic Studies to Evaluate the Effect of Nod-Factor-Based Fertilizer on the Maize (Zea mays) Stem.

Maize (Zea mays) is one of the most cultivated plants in the world. Due to the large area, the scale of its production, and the demand to increase the yield, there is a need for new environmentally friendly fertilizers. One group of such candidates is bacteria-produced nodulation (or nod) factors. Limited research has explored the impact of nodulation, factors on maize within field conditions, with most studies restricted to greenhouse settings and early developmental stages. Additionally, there is a scarcity of investigations that elucidate the metabolic alterations in the maize stem due to nod-factor exposure. It was therefore the aim of this study. Maize stem's metabolites and fibers were analyzed with various imaging analytical techniques: matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), and diffuse reflectance infrared Fourier transform spectroscopy. Moreover, the biochemical analyses were used to evaluate the proteins and soluble carbohydrates concentration and total phenolic content. These techniques were used to evaluate the influence of nod factor-based biofertilizer on the growth of a non-symbiotic plant, maize. The biofertilizer increased the grain yield and the stem mass. Moreover, the spectroscopic and biochemical investigation proved the appreciable biochemical changes in the stems of the maize in biofertilizer-treated plants. Noticeable changes were found in the spatial distribution and the increase in the concentration of flavonoids such as maysin, quercetin, and rutin. Moreover, the concentration of cell wall components (fibers) increased. Furthermore, it was shown that the use of untargeted analyses (such as Raman and ATR FT-IR, spectroscopic imaging, and MALDI-MSI) is useful for the investigation of the biochemical changes in plants.

Utilizing multimodal mass spectrometry imaging for profiling immune cell composition and N-glycosylation across colorectal carcinoma disease progression.

Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.

Unraveling spatial metabolome of the aerial and underground parts of Scutellaria baicalensis by matrix-assisted laser desorption/ionization mass spectrometry imaging

BackgroundScutellaria baicalensis Georgi, a traditional Chinese medicine, is clinically applied mainly as the dried root of Scutellaria baicalensis, and the aerial parts of Scutellaria baicalensis, its stems and leaves, are often consumed as "Scutellaria baicalensis tea" to clear heat, dry dampness, reduce fire and detoxify, while few comparative analyses of the spatial metabolome of the aerial and underground parts of Scutellaria baicalensis have been carried out in current research. MethodsIn this work, Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize the spatial imaging of the root, stem, and leaf of Scutellaria baicalensis at a high resolution of 10 μm, respectively, investigating the spatial distribution of the different secondary metabolites in the aerial and underground parts of Scutellaria baicalensis. ResultsIn the present results, various metabolites, such as flavonoid glycosides, flavonoid metabolites, and phenolic acids, were systematically characterized in Scutellaria baicalensis root, stem, and leaf. Nine glycosides, 18 flavonoids, one organic acid, and four other metabolites in Scutellaria baicalensis root; nine glycosides, nine flavonoids, one organic acid in Scutellaria baicalensis stem; and seven flavonoids and seven glycosides in Scutellaria baicalensis leaf were visualized by MALDI-MSI. In the underground part of Scutellaria baicalensis, baicalein, wogonin, baicalin, wogonoside, and chrysin were widely distributed, while there was less spatial location in the aerial parts. Moreover, scutellarein, carthamidin/isocarthamidin, scutellarin, carthamidin/isocarthamidin-7-O-glucuronide had a high distribution in the aerial parts of Scutellaria baicalensis. In addition, the biosynthetic pathways involved in the biosynthesis of significant flavonoid metabolites in aerial and underground parts of Scutellaria baicalensis were successfully localized and visualized. ConclusionsMALDI-MSI offers a favorable approach for investigating the spatial distribution and effective utilization of metabolites of Scutellaria baicalensis. The detailed spatial chemical information can not only improve our understanding of the biosynthesis pathways of flavonoid metabolites, but more importantly, suggest that we need to fully exert the overall medicinal value of Scutellaria baicalensis, strengthening the reuse and development of the resources of Scutellaria baicalensis aboveground parts.

B4galt1 Regulates the WNT-β-Catenin Axis to Control Hematopoietic Stem and Progenitor Cells (HSPCs) Fitness

In achieving the optimal numbers of fully functional blood cells across different lineages, intrinsic developmental programs within the cells and external guidance mechanisms are crucial. Glycans on the cell surface and extracellular environment are pivotal in regulating cell maintenance, differentiation, and function. We recently discovered that the glycosyltransferase β-1,4-galactosyltransferase 1 (B4galt1) leads to dysplastic megakaryocytes, impaired thrombopoiesis, and increased hematopoietic stem cells (HSC). To investigate the relationship between B4galt-dependent glycosylation and HSC function, we analyzed the glycan composition in control and B4galt1 null (B4 -/-) femurs by spatial mass spectrometry using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). In control femurs, we observed a distinct gradient of complex N-glycans, with higher expression at the distal ends and decreasing complexity towards the shaft, showcasing diverse glycan expression in the hematopoietic environment. However, this gradient was absent in B4 -/-femurs, where we detected an increase of immature N-glycans and a concurrent decrease in complex N-glycan structures (p&amp;lt;0.05). Additionally, we observed aberrant O-glycosylation in B4 -/- LT-HSCs, as determined using lectin arrays. The glycan composition in the absence of B4galt1-dependent glycosylation mirrors cancer-associated glycan patterns. To further understand the role of B4galt1 in steady-state hematopoiesis, we next examined the bone marrow (BM) immunophenotypic composition of B4 -/- mice. Flow cytometry analysis revealed a significant expansion of immunophenotypically defined long term (LT)-HSCs (p=0.0061) and multipotent progenitors (MPPs) (p&amp;lt;0.05) in B4 -/-BM. Additionally, B4 -/- LT-HSCs showed higher expression of the platelet marker CD41 (p=0.0171), suggesting the presence of an expanded megakaryocyte (MK) biased LT-HSCs population (LT-HSC CD41+ cells). This expansion occurred without pro-inflammatory cues, as determined through cytokine arrays. To investigate if the global transcriptional landscape in B4 -/- cells could explain the observed expansion phenotype, we conducted droplet-based single-cell RNA-seq. Transfer learning classification allowed us to identify an expansion of the transcriptional output associated with LT-HSC and MPP2 signatures in B4 -/- cells (p&amp;lt;0.05). Further transcriptional analysis revealed an increased number of B4 -/- cells expressing MKs specific markers (CD41, Pf4, GP9, Vwf, GP1ba), providing additional support for the expansion of B4 -/- MK-biased LT-HSCs. Gene Set Enrichment Analysis (GSEA) of B4 -/- LT-HSCs demonstrated enrichment in metabolism-associated pathways and cell cycle regulation (FDR&amp;lt;0.05). Conversely, critical differentiation processes at the stem cell level, such as cellular adhesion and lineage commitment, were downregulated (FDR&amp;lt;0.05). To investigate the role of B4galt1 in HSC expansion, we transcriptionally classified LT-HSCs based on their cell cycle status (G1, G2M, and S). Analysis of LT-HSC CD41+ cell cycle distribution revealed an increased in S phase in B4 -/- LT-HSC CD41+ cells. Additionally, B4 -/- LT-HSC CD41+ cells exhibited enrichment in cell cycle regulation pathways, including DNA replication, DNA strand elongation, and G1 to S cell cycle control (FDR&amp;lt;0.05). Conversely, critical pathways associated with HSC homeostasis and quiescence were depleted in the absence of B4galt1 (FDR&amp;lt;0.05). GSEA analysis unveiled upregulation of Wnt and Myc pathways in B4 -/- LT-HSC CD41+ cells. Immunofluorescence and immunoblot analysis demonstrated elevated total β-catenin levels in B4 -/- LSK cells and enhanced nuclear translocation of non-phosphorylated Ser33/37/Thr41 β-catenin. To confirm the functional significance, we treated B4 -/- LSK cells with the Wnt inhibitor XAV939 which normalized cell numbers to control levels and cause a reduction in the observed expansion at the stem and progenitor compartments. These findings suggest that B4galt1 is vital in regulating HSC expansion, likely through the Wnt/β-catenin signaling pathway. Our data provide compelling evidence that B4galt1 regulates steady-state hematopoiesis, through its influence on glycosylation in the bone marrow niche, highlighting the critical role of the B4galt1-Wnt-β-catenin axis in regulating LT-HSC fitness and expansion.

Spatiotemporal visualization of the synthesis and accumulation of rotenone in Derris elliptica roots using mass spectrometry imaging

The discovery of novel botanical pesticides as a preferred alternative to synthetic pesticides is regarded as an environmentally friendly strategy, yet it remains a great challenge due to limited insights into the synthesis and accumulation of active ingredient intermediates. Herein, we demonstrate the use of gold nanoparticle (AuNP)-assisted laser desorption/ionization mass spectrometry imaging (LDI-MSI) for the tissue-specific distribution and spatiotemporal accumulation effect of rotenone and active ingredient intermediates in its biosynthetic pathway within the roots of Derris elliptica. The MSI results revealed that rotenone was mainly concentrated in the epidermis, cortex, and xylem in the first two years and began to accumulate in the phloem since the 3rd year. Meanwhile, the rotenone contents increased in the epidermis, cortex, and xylem in the 4th year and finally decreased in the 5th year. The ultra-performance liquid chromatography (UPLC) results revealed that there was a significant difference in rotenone content between the root and non-root bark (p < 0.05) except for the 1st year. It reached its maximum value at 9.71% in the root bark in the 4th year and 7.98% in the non-root bark in the 1st year, which was in accordance with the MSI results. Additionally, the active ingredient intermediates in the Hydroxylation/Methylation and Rotenoid Phases of the biosynthetic pathway of rotenone were concentrated in the root region. Taken together, the collective results provide the scientific guidance for the scientific cultivation of D. elliptica and the efficient extraction of botanical pesticides.

Detection of Distinct Distributions of Acetaminophen and Acetaminophen-Cysteine in Kidneys up to 10 μm Resolution and Identification of a Novel Acetaminophen Metabolite Using an AP-MALDI Imaging Mass Microscope.

Drug distribution studies in tissue are crucial for understanding the pharmacokinetics and potential toxicity of drugs. Recently, matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has gained attention for drug distribution studies due to its high sensitivity, label-free nature, and ability to distinguish between parent drugs, their metabolites, and endogenous molecules. Despite these advantages, achieving high spatial resolution in drug imaging is challenging. Importantly, many drugs and metabolites are rarely detectable by conventional vacuum MALDI-MSI because of their poor ionization efficiency. It has been reported that acetaminophen (APAP) and one of its major metabolites, APAP-Cysteine (APAP-CYS), cannot be detected by vacuum MALDI-MSI without derivatization. In this context, we showed the distribution of both APAP and APAP-CYS in kidneys at high spatial resolution (25 and 10 μm) by employing an atmospheric pressure-MALDI imaging mass microscope without derivatization. APAP was highly accumulated in the renal pelvis 1 h after drug administration, while APAP-CYS exhibited characteristic distributions in the outer medulla and renal pelvis at both 30 min and 1 h after administration. Interestingly, cluster-like distributions of APAP and APAP-CYS were observed in the renal pelvis at 10 μm spatial resolution. Additionally, a novel APAP metabolite, tentatively coined as APAP-butyl sulfate (APAP-BS), was identified in the kidney, brain, and liver by combining MSI and tandem MSI. For the first time, our study revealed differential distributions of APAP, APAP-CYS (in kidneys), and APAP-BS (in kidney, brain, and liver) and is believed to enhance the understanding of the pharmacokinetics and potential nephrotoxicity of this drug.

A sensitive on-tissue chemical derivatization-mass spectrometry imaging method for the quantitative visualization of helicid in mice

Helicid (4-formylphenyl-O-β-d-allopyranoside) is an aromatic phenolic glucoside. It is one of the main active constituents from the seeds of the Chinese herb Helicia nilagirica. Helicid has been reported to exert sedative, analgesic, hypnotic and antidepressant effects, and is currently used clinically to treat neurasthenic syndrome, vascular headaches and trigeminal neuralgia. On-tissue chemical derivatization (OTCD) combined with matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was developed for the quantitative visualization of helicid in mice. The derivatization reagent, Girard’s reagent T (GT), was sprayed directly onto the tissue surface using an automated pneumatic TM-sprayer. The OTCD reaction was conducted in a closed container with 120 mL of 50% methanol with 0.4% TFA at 40 °C for 40 min. α-Cyano-4-hydroxycinnamic acid (2.22 μg/mm2) was used as the matrix, which was deposited onto the tissue surface. MALDI-MSI analysis was performed in the positive-ion mode with a spatial resolution of 200 μm. The sensitivity for the MALDI-MSI of helicid in tissue sections increased 1920 times after derivatization. The assay showed good linearity in the helicid concentration range of 0.32–32 ng/mm2. The method was used to investigate the spatiotemporal distribution of helicid in mice.

Monoisotopic silver nanoparticles-based mass spectrometry imaging of human bladder cancer tissue: Biomarker discovery

PurposeBladder cancer (BC) is the 10th most common form of cancer worldwide and the 2nd most common cancer of the urinary tract after prostate cancer, taking into account both incidence and prevalence. Materials/methodsTissues from patients with BC and also tissue extracts were analyzed by laser desorption/ionization mass spectrometry imaging (LDI-MSI) with monoisotopic silver-109 nanoparticles-enhanced target (109AgNPET). ResultsUnivariate and multivariate statistical analyses revealed 10 metabolites that differentiated between tumor and normal tissues from six patients with diagnosed BC. Selected metabolites are discussed in detail in relation to their mass spectrometry (MS) imaging results. The pathway analysis enabled us to link these compounds with 17 metabolic pathways. ConclusionsAccording to receiver operating characteristic (ROC) analysis of biomarkers, 10 known metabolites were identified as the new potential biomarkers with areas under the curve (AUC) higher than >0.99. In both univariate and multivariate analysis, it was predicted that these compounds could serve as useful discriminators of cancerous versus normal tissue in patients diagnosed with BC.

Exploring Bacterial Resistant Metabolism by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging

The inappropriate and excessive use of antibiotics for the treatment of bacterial infections has led to the increasing presence of resistant and multidrug-resistant bacteria both in hospital settings and in the community. Thus, understanding the metabolism of resistant bacteria is extremely important to combat them more efficiently. In this scenario, mass spectrometry imaging (MSI) is considered a promising technique for understanding the resistant characteristics of such bacteria and how they can potentially be treated. This process consists of the identification of different ions on the surface of the colonies and the identification of potential metabolites that characterize antibiotic resistance, upon comparison with susceptible bacteria of the same species. This work presents matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) study of colonies of Methicillin-resistant Staphylococcus aureus, as a proof of concept of the technique for obtaining images of bacteria colonies. Images of methicillin-resistant and susceptible colonies of Staphylococcus aureus were obtained by a sublimation process to apply the MALDI matrix on the samples followed by MALDI-MSI analysis. Seventeen (17) potential metabolites were identified and spatially localized, such as N,N-dihydroxy-L-valine, 2-(4-Methylphenyl)ethylamine, 3,4-Dihydroxy-L-phenylalanine, 2-Methyl-hexanoic acid, threonine, Arginine, Aureusimine and Glycyl-D-asparagine. Thus, this study reinforces the potential of MALDI-MSI for identification of metabolites synthesized by different strains of Staphylococcus aureus bacteria.

Spatially resolved metabolomics reveals variety-specific metabolic changes in banana pulp during postharvest senescence

Banana is one of most popular fruits globally due to health-promoting and disease-preventing effects, yet little is known about in situ metabolic changes across banana varieties. Here, we integrated gold nanoparticle (AuNP)-assisted laser desorption/ionization mass spectrometry imaging (LDI-MSI) and metabolomics to investigate the spatiotemporal distribution and levels of metabolites within Brazil and Dongguan banana pulps during postharvest senescence. Metabolomics results indicated that both postripening stages and banana varieties contribute to metabolite levels. Benefiting from improved ionization efficiency of small-molecule metabolites and less peak interference, we visualized the spatiotemporal distribution of sugars, amino acids (AAs) and monoamines within pulps using AuNP-assisted LDI-MSI for the first time, revealing that AAs and monoamines exclusively accumulated in the middle region near the seed zone. Monosaccharides and di/trisaccharides were generally distributed across entire pulps but exhibited different accumulation patterns. These findings provide a guide for breeding new varieties and improving extraction efficiency of bioactive compounds.

Tissue Imprinting on 2D Nanoflakes-Capped Silicon Nanowires for Lipidomic Mass Spectrometry Imaging and Cancer Diagnosis.

Spatially resolved tissue lipidomics is essential for accurate intraoperative and postoperative cancer diagnosis by revealing molecular information in the tumor microenvironment. Matrix-free laser desorption ionization mass spectrometry imaging (LDI-MSI) is an emerging attractive technology for label-free visualization of metabolites distributions in biological specimens. However, the development of LDI-MSI technology that could conveniently and authentically reveal molecular distribution on tissue samples is still a challenge. Herein, we present a tissue imprinting technology by retaining tissue lipids on 2D nanoflakes-capped silicon nanowires (SiNWs) for further mass spectrometry imaging and cancer diagnosis. The 2D nanoflakes were prepared by liquid exfoliation of molybdenum disulfide (MoS2) with nitrogen-doped graphene quantum dots (NGQDs), which serve as both intercalation agent and dispersant. The obtained NGQD@MoS2 nanoflakes were then decorated on the tip of vertical SiNWs, forming a hybrid NGQD@MoS2/SiNWs nanostructure, which display excellent lipid extraction ability, enhanced LDI efficiency and molecule imaging capability. The peak number and total ion intensity of different lipids species on animal lung tissues obtained by tissue imprinting LDI-MSI on NGQD@MoS2/SiNWs were ∼4-5 times greater than those on SiNWs substrate. As a proof-of-concept demonstration, the NGQD@MoS2/SiNWs nanostructure was further applied to visualize phospholipids on sliced non small cell lung cancer (NSCLC) tissue along with the adjacent normal tissue. On the basis of selected feature lipids and machine learning algorithm, a prediction model was constructed to discriminate NSCLC tissues from the adjacent normal tissues with an accuracy of 100% for the discovery cohort and 91.7% for the independent validation cohort.

Cytomolecular Classification of Thyroid Nodules Using Fine-Needle Washes Aspiration Biopsies.

Fine-needle aspiration biopsies (FNA) represent the gold standard to exclude the malignant nature of thyroid nodules. After cytomorphology, 20–30% of cases are deemed “indeterminate for malignancy” and undergo surgery. However, after thyroidectomy, 70–80% of these nodules are benign. The identification of tools for improving FNA’s diagnostic performances is explored by matrix-assisted laser-desorption ionization mass spectrometry imaging (MALDI-MSI). A clinical study was conducted in order to build a classification model for the characterization of thyroid nodules on a large cohort of 240 samples, showing that MALDI-MSI can be effective in separating areas with benign/malignant cells. The model had optimal performances in the internal validation set (n = 70), with 100.0% (95% CI = 83.2–100.0%) sensitivity and 96.0% (95% CI = 86.3–99.5%) specificity. The external validation (n = 170) showed a specificity of 82.9% (95% CI = 74.3–89.5%) and a sensitivity of 43.1% (95% CI = 30.9–56.0%). The performance of the model was hampered in the presence of poor and/or noisy spectra. Consequently, restricting the evaluation to the subset of FNAs with adequate cellularity, sensitivity improved up to 76.5% (95% CI = 58.8–89.3). Results also suggest the putative role of MALDI-MSI in routine clinical triage, with a three levels diagnostic classification that accounts for an indeterminate gray zone of nodules requiring a strict follow-up.

Laser Desorption/Ionization Mass Spectrometry Imaging: A New Tool to See through Nanoscale Particles in Biological Systems.

Understanding the fate of nanoscale particles (NPs) in biological systems is significant with the increasing risk for human exposure. Recent research endeavors in laser desorption/ionization mass spectrometry imaging (LDI-MSI) have enriched the toolbox for evaluation of NPs' behavior in biological tissues, especially in aspects including sub-organ bio-distribution, clearance, quantification and surface chemistry variation analysis. In recognition of the potential for advancement in LDI MSI, this concept provides a brief overview of recent research works in LDI MSI for NPs, illustrates new applications that demonstrate the superiority of this technique, and highlights a series of perspectives and directions to move the field forward.

Proteomic Distributions in CD34+ Microvascular Niche Patterns of Glioblastoma.

The poor clinical prognosis and microvascular patterns of glioblastoma (GBM) are of serious concern to many clinicians and researchers. However, very few studies have examined the correlation between microvascular niche patterns (MVNPs) and proteomic distribution. In this study, CD34 immunofluorescence staining and matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-IMS) technology were used to investigate the protein distributions in MVNPs. CD34+ microvascular phenotype could be divided into four types: microvascular sprouting (MS), vascular cluster (VC), vascular garland (VG), and glomeruloid vascular proliferation (GVP). Based on such characteristics, MVNPs were divided into two types by cluster analysis, namely, type I, comprising primarily MS and VC, and type II, comprising many VGs and GVPs. Survival analysis indicated the type of MVNPs to be an independent prognostic factor for progression-free and overall survival in GBM. MALDI-IMS results showed the peaks at m/z 1037 and 8960 to exhibit stronger ion signals in type II, while those at m/z 3240 and 3265 exhibited stronger ion signals in type I. The findings may assist future research on therapy and help predict prognosis in GBM. However, due to the limited number of studies, more well-designed studies are warranted to further verify our results.

Abstract 2819: Mass spectrometry imaging reveals heterogeneous glycogen metabolism in non small cell lung cancer

Abstract Lung cancer is the leading cause of cancer related death worldwide, with a high mortality rate even when diagnosed at an early stage. Identifying the unique molecular features that drive disease progression within distinct tumor subtypes is a critical step in lung cancer research that could advance our understanding of lung cancer biology, leading to predictive biomarkers and novel therapies. Glycogen is the primary source of carbohydrate storage in most issues, and its degradation products are intimately connection to central carbon metabolism. Recently, aberrant glycogen accumulation has been observed in lung tumors and been reported to promote lung cancer progression. Despite these findings, the full clinical implications of dysregulated glycogen metabolism and its role in lung cancer tumorigenesis remain largely unknown. Due to its unique physiochemical properties, glycogen is extremely difficult to measure biochemically and visualize in situ. Herein, we describe a novel method to image microenvironmental glycogen via enzymatic release of glycogen substrates coupled to matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). Our assay provides robust information on glycogen structure and phosphate composition with spatial distribution. With this technique, we analyzed glycogen chain length and glycogen phosphate content from tissue microarrays containing over 200 lung cancer patient samples banked at the University of Kentucky with over 10 years of clinical metadata. Using MALDI-MSI, we found significant glycogen accumulation in lung tumor tissue compared to match normal lung across all patients, and found adenocarcinoma (LUAD) samples contained higher glycogen content than squamous cell carcinoma (SCC). Regional analysis by MALDI-MSI revealed SCC primarily accumulates glycogen in the stroma and blood vessels, but not in tumors. Notably, LUAD patient tumors exhibit decreased expression of laforin, a glucan phosphatase that plays a key role in glycogen phosphorylation and glycogen degradation. Low laforin expression also corresponds to poorer overall survival in LUAD patients. In vivo, deletion of Epm2a (the gene that encodes laforin) in KRASG12D/p53-/- mice results in increased tumor burden and accumulation of hyperphosphorylated intratumoral glycogen. Further, A549 LUAD xenografts treated with guaiacol, a glycogen synthase inhibitor, show markedly reduced tumor growth, while guaiacol had no effect on H2030 SCC tumor growth. Given the significant effect of glycogen accumulation on in vivo tumor growth, our data suggest the glycogen metabolic pathway represents a novel therapeutic target specifically for LUAD patients. Together, our study describes a novel method to visualize glycogen structure using MALDI-MSI and reveals unique metabolic features that contribute to LUAD disease progression. Citation Format: Lindsey R. Conroy, Lyndsay EA Young, Harrison A. Clarke, Alexandra E. Stanback, Michael D. Buoncristiani, Christine F. Brainson, Derek B. Allison, Ramon C. Sun, Richard R. Drake. Mass spectrometry imaging reveals heterogeneous glycogen metabolism in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2819.

In Situ Analysis of N-Linked Glycans as Potential Biomarkers of Clinical Course in Human Prostate Cancer.

Prostate cancer is the most common cancer in men worldwide. Despite its prevalence, there is a critical knowledge gap in understanding factors driving disparities in survival among different cohorts of patients with prostate cancer. Identifying molecular features separating disparate populations is an important first step in prostate cancer research that could lead to fundamental hypotheses in prostate biology, predictive biomarker discovery, and personalized therapy. N-linked glycosylation is a cotranslational event during protein folding that modulates a myriad of cellular processes. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Herein, we performed direct on-tissue analysis of N-linked glycans using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) from tissue microarrays of over 100 patient tumors with over 10 years of follow-up metadata. We successfully identified a panel of N-glycans that are unique between benign and prostate tumor tissue. Specifically, high-mannose as well as tri-and tetra-antennary N-glycans were more abundant in tumor tissue and increase proportionally with tumor grade. Further, we expanded our analyses to examine the N-glycan profiles of Black and Appalachian patients and have identified unique glycan signatures that correlate with recurrence in each population. Our study highlights the potential applications of MALDI-MSI for digital pathology and biomarker discovery for prostate cancer. IMPLICATIONS: MALDI-MSI identifies N-glycan perturbations in prostate tumors compared with benign tissue. This method can be utilized to predict prostate cancer recurrence and study prostate cancer disparities.

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging by Freeze-Spot Deposition of the Matrix.

Imaging mass spectrometry has emerged as a powerful metabolite measurement approach to capture the spatial dimension of metabolite distribution in a biological sample. In matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), deposition of the chemical-matrix onto the sample serves to simultaneously extract biomolecules to the sample surface and concurrently render the sample amenable to MALDI. However, matrix application may mobilize sample metabolites and will dictate the efficiency of matrix crystallization, together limiting the lateral resolution which may be optimally achieved by MSI. Here, we describe a matrix application technique, herein referred to as the "freeze-spot" method, conceived as a low-cost preparative approach requiring minimal amounts of chemical matrix while maintaining the spatial dimension of sample metabolites for MALDI-MSI. Matrix deposition was achieved by pipette spot application of the matrix-solubilized within a solvent solution with a freezing point above that of a chilled sample stage to which the sample section is mounted. The matrix solution freezes on contact with the sample and the solvent is removed by sublimation, leaving a fine crystalline matrix on the sample surface. Freeze-spotting is quick to perform, found particularly useful for MALDI-MSI of small sample sections, and well suited to efficient and cost-effective method development pipelines, while capable of maintaining the lateral resolution required by MSI.

Brain Lipid Dynamics in Amyloid Precursor Protein/Presenilin 1 Mouse Model of Early Alzheimer's Disease by Desorption Electrospray Ionization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging Techniques.

Alzheimer's disease (AD) is closely associated with lipid metabolism dysfunction. However, space distribution and metabolism of aberrant lipids in the brain of early-stage AD mouse remain unclear. In our current work, a novel lipidomics method based on mass spectrometry imaging was developed to visually disclose molecular perturbation and characterize space distribution in the brain of double transgenic amyloid precursor protein/presenilin 1 mouse (2 and 3 months old). Significant changes were detected, including phosphatidylethanolamines, phosphatidylcholines, fatty acids, lysophospholipids, and glycerides in AD mouse brain. The results in this study suggest that these significantly altered lipid metabolic pathways (glycerophospholipid metabolism) may be implicated in early-stage AD. Our work deepens the understanding of the physio-pathologic mechanism of early-stage AD.

A Near-Infrared-II Polymer with Tandem Fluorophores Demonstrates Superior Biodegradability for Simultaneous Drug Tracking and Treatment Efficacy Feedback.

NIR-II (1000-1700 nm) fluorescence imaging is continually attracting strong research interest. However, current NIR-II imaging materials are limited to small molecules with fast blood clearance and inorganic nanomaterials and organic conjugated polymers of poor biodegradability and low biocompatibility. Here, we report a highly biodegradable polyester carrying tandem NIR-II fluorophores as a promising alternative. The polymer encapsulated a platinum intercalator (56MESS, (5,6-dimethyl-1,10-phenanthroline) (1S,2S-diaminocyclohexane) platinum(II)) and was conjugated with both a cell-targeting RGD peptide and a caspase-3 cleavable peptide probe to form nanoparticles for simultaneous NIR-II and apoptosis imaging. In vitro, the nanoparticles were approximately 4-1000- and 1.5-10-fold more potent than cisplatin and 56MESS, respectively. Moreover, in vivo, they significantly inhibited tumor growth on a multidrug-resistant patient-derived mouse model (PDXMDR). Finally, through label-free laser desorption-ionization mass spectrometry imaging (MALDI-MSI), in situ 56MESS release in the deeper tumors was observed. This work highlighted the use of biodegradable NIR-II polymers for monitoring drugs in vivo and therapeutic effect feedback in real-time.