Tissue Imaging Mass Spectrometry Research Articles

The simultaneous inhibition of SLC6A19 and BCRP transporters leads to an increase of indoxyl sulfate (a uremic toxin) in plasma and kidney.

SLC6A19 inhibitors are being studied as therapeutic agents for Phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7{plus minus}7.7 compared to vehicle 0.3{plus minus}0.1 (P=0.01) as determined by tissue mass spectrometry imaging (tMSI) analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters OAT1, OAT3 and BCRP. RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using LC-MS. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, while compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. Significance Statement This is the first publication to decipher the mechanism for accumulation of IDS (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the GI track increases IDS formation and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both SLC6A19 and BCRP simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.

Kidney injury contributes to edema of zebrafish larvae caused by quantum dots

Edema represents a notable outcome in fishes exposed to aquatic pollutants, yet the underlying etiology remains inadequately understood. This investigation delves into the etiological factors of edema formation in 7 days post fertilization (dpf) zebrafish larvae following their exposure to InP/ZnS quantum dots (QDs), which was chosen as a prototypical edema inducer. Given the fundamental role of the kidney in osmoregulation, we used transgenic zebrafish lines featuring fluorescent protein labeling of the glomerulus, renal tubule, and blood vessels, in conjunction with histopathological scrutiny. We identified the pronounced morphological and structural aberrations within the pronephros. By means of tissue mass spectrometry imaging and hyperspectral microscopy, we discerned the accumulation of InP/ZnS QDs in the pronephros. Moreover, InP/ZnS QDs impeded the renal clearance capacity of the pronephros, as substantiated by diminished uptake of FITC-dextran. InP/ZnS QDs also disturbed the expression levels of marker genes associated with kidney development and osmoregulatory function at the earlier time points, which preceded the onset of edema. These results suggest that impaired fluid clearance most likely resulting from pronephros injury contributes to the emergence of zebrafish edema. Briefly, our study provides a perspective: the kidney developmental injury induced by exogenous substances may regulate edema in a zebrafish model.

OGC O09 The clinical utilisation of tissue imaging Mass Spectrometry as a diagnostic model for Oesophageal Adenocarcinoma

Abstract Background The prevalence of Oesophageal adenocarcinoma (OA) in the United Kingdom has steadily risen since the 1970's. The majority of patients present with advanced disease. Early diagnosis utilising point of care testing may enable an increase in the number of patients managed on a potentially curative pathway. Tissue Imaging Mass Spectrometry by Desorption Electrospray (DESI-MS) is one such tool to differentiate the abundance of lipids in OA. This work presents the novel use of Mass Spectrometry (MS) imaging to identify variations in lipid abundance between OA and normal oesophagus (NO). Methods Tissue was sampled from specimens dissected immediately post-resection from the tumour and at 5-centimetre intervals in the proximal NO. Forty-one unique patient OA and NO tissue samples were analysed in triplicate by DESI-MS tissue imaging to differentiate Glycerophospholipids. A total of 246 DESI-MS images were correlated with paired formalin fixed Haematoxylin and Eosin stained tissue slides, the current gold standard cancer diagnostic test. Principle component analysis utilising a supervised model classified tissue types with a significance of p=0.01. Receiver operator curves (ROC) were used to determine the diagnostic sensitivity and specificity of DESI-MS for the identification of adenocarcinoma. Results Tissue analysis confirmed unsaturated Phosphatidylglycerols and Phosphatidic acids were significantly more abundant in OA compared to NO (p<0.001). Tissue analysis by a Partial Least Square (PLS) supervised linear regression model classified cancer and non-cancer tissue with a confusion matrix classification of 93.3% and 70.8% for OA and NO (Figure 1a-c). A diagnostic sensitivity of 85% and specificity of 88% was determined by ROC curve. The abundance of Phosphatidylglycerol and Phosphatidylserine decreased proportionally in analysed tissue sections at 5cm and 10cm from the primary tumour, as did the degree of lipid unsaturation. Conclusions This work presents a novel tissue imaging MS model to differentiate lipid abundance in OA. An abundance ratio of 1.17:1 (OA:NO) of unsaturated Phosphatidylglycerol was diagnostic of cancer by DESI-MS with a high sensitivity and specificity. This MS tissue analysis model has the potential to enable accurate point of care diagnostics for OA. In addition, we suggest oesophageal cancer induced perturbation of Glycerophospholipid metabolism may be a potential target for future chemotherapeutics.

Abstract B019: Discrete regulation of the collagen proteome among pathological features in DCIS and invasive breast cancer by mass spectrometry tissue imaging

Abstract Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). In recent publications, collagen stromal differences have been reported between patients that progressed to IBC (progressors) and those that did not (non-progressors). However, details on the role spatial regulation of the collagen proteome might play in this progression have yet to be studied. Here, we investigated the pathological distribution of collagen post-translational modifications in a cohort of patients classified as progressors with ipsilateral IBC recurrence compared to non-progressors. Previously published methods for collagen proteomics by targeted tissue mass spectrometry imaging were used. The method reports collagen types and post-translational modifications within the collagen triple-helical region as well as approximately 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Initial studies investigated collagen variation in lumpectomies (n=7) with DCIS, DCIS plus invasive ductal carcinoma (IDC) or IDC only. Over 590 peptides were found to be linked to annotated pathologies. A preliminary comparison of DCIS (n=2392 spectra) to IBC (n=4696 spectra) using area under the receiver operating curve (AUROC) ≥0.85 demonstrated that 47 peptides could individually discriminate between DCIS and IBC in this limited cohort. Image segmentation of the 405,652 pixels demonstrated 11 high-level hierarchical groups designating unique spatially localized ECM proteomic groups; these groups overlaid with histopathological features and pathological annotations. A total of 87 samples from the Resource of Archival Breast Tissue (RAHBT) matched with clinical characteristics were also investigated. Cores were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. Initial results suggest certain peptides may differentiate between non-progressors and progressors with ipsilateral IBC recurrence. Our current work focuses on correlating collagen signatures to mixed pathologies and the cellular content of cores. Further investigation of the collagen proteome is warranted. Overall, the data suggest unique collagen signatures in DCIS that could be useful for understanding recurrence and progression to IBC. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Robert Michael Angelo, Graham Colditz, Eun-Sil Shelley Hwang, Robert West, Laura Spruill, Jeffrey R. Marks, Richard R. Drake, Peggi M. Angel. Discrete regulation of the collagen proteome among pathological features in DCIS and invasive breast cancer by mass spectrometry tissue imaging [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B019.

Next-Generation Cancer Magnetic Resonance Imaging With Tumor-Targeted Alkylphosphocholine Metal Analogs.

In an effort to exploit the elevated need for phospholipids displayed by cancer cells relative to normal cells, we have developed tumor-targeted alkylphosphocholines (APCs) as broad-spectrum cancer imaging and therapy agents. Radioactive APC analogs have exhibited selective uptake and prolonged tumor retention in over 50 cancer types in preclinical models, as well as over 15 cancer types in over a dozen clinical trials. To push the structural limits of this platform, we recently added a chelating moiety capable of binding gadolinium and many other metals for cancer-targeted magnetic resonance imaging (MRI), positron emission tomography imaging, and targeted radionuclide therapy. The aim of this work was to synthesize, characterize, and validate the tumor selectivity of a new broad-spectrum, tumor-targeted, macrocyclic MRI chelate, Gd-NM600, in xenograft and orthotopic tumor models. A secondary aim was to identify and track the in vivo chemical speciation and spatial localization of this new chelate Gd-NM600 in order to assess its Gd deposition properties. T1 relaxivities of Gd-NM600 were characterized in water and plasma at 1.5 T and 3.0 T. Tumor uptake and subcellular localization studies were performed using transmission electron microscopy. We imaged 8 different preclinical models of human cancer over time and compared the T1-weighted imaging results to that of a commercial macrocyclic Gd chelate, Gd-DOTA. Finally, matrix-assisted laser desorption and ionization-mass spectrometry imaging was used to characterize and map the tissue distribution of the chemical species of Gd-NM600. Gd-NM600 exhibits high T1 relaxivity (approximately 16.4 s-1/mM at 1.5 T), excellent tumor uptake (3.95 %ID/g at 48 hours), prolonged tumor retention (7 days), and MRI conspicuity. Moreover, minimal tumor uptake saturability of Gd-NM600 was observed. Broad-spectrum tumor-specific uptake was demonstrated in 8 different human cancer models. Cancer cell uptake of Gd-NM600 via endosomal internalization and processing was revealed with transmission electron microscopy. Importantly, tissue mass spectrometry imaging successfully interrogated the spatial localization and chemical speciation of Gd compounds and also identified breakdown products of Gd species. We have introduced a new macrocyclic cancer-targeted Gd chelate that achieves broad-spectrum tumor uptake and prolonged retention. Furthermore, we have demonstrated in vivo stability of Gd-NM600 by ultrahigh resolution MS tissue imaging. A tumor-targeted contrast agent coupled with the enhanced imaging resolution of MRI relative to positron emission tomography may transform oncologic imaging.

Abstract P1-10-05: Evidence that body mass index modifies breast tissue collagen peptide response pattern to treatment with the non-steroidal anti-inflammatory drug sulindac

Abstract Background. In observational studies, regular use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced breast cancer risk and disease recurrence with evidence that the benefit is greater in overweight/obese women. This parallels findings that obesity is associated with an increase in proinflammatory processes with possible effects on the breast stroma and extracellular matrix. Methods. To assess if body mass index (BMI kg/m2) alters breast tissue response to NSAIDs, we conducted exploratory analyses of change in breast tissue collagen-associated peptides following 6 months of treatment with the NSAID sulindac (150 mg bid) by BMI category: normal (18.5-24.9 n=6), overweight (25-29.9 n=14) and obese (≥30 n=11). Samples for this study were non-cancer core needle breast biopsies from postmenopausal women with a history of hormone receptor positive breast cancer enrolled to study sulindac effect on breast tissue biomarkers. At completion, 36 of 50 patients underwent baseline biopsy and 31 had sufficient tissue for paired analyses. Tissue collagen-associated peptides were studied using whole slide tissue imaging mass spectrometry proteomics. Individual peptide signals were normalized to total ion current and mean peak intensity per area across the entire biological specimen were used to generate a score per patient. Change in stroma peptides was evaluated by BMI status using the Wilcoxon matched pairs signed rank test. Unsupervised hierarchical clustering and heatmap visualization were used to assess differential expression of the peaks. Results. Approximately, 550 peptide peaks were found by targeted collagen tissue imaging proteomics. Striking differences in response to sulindac were observed by BMI. In overweight patients, 6 peptides related to COL1A1, COL6A1, COL6A3 and VIM on database matching differed significantly before and after sulindac treatment. Three were also altered in obese women. Two of the peptides overlapped 10 peptide changes identified in unstratified analyses reported in a separate submitted abstract on the main effect of sulindac on tissue collagen. Interestingly, these peptides were not altered in patients with normal BMI. In addition, on study 15 patients experienced a decrease in BMI and 12 experienced an increase. Two collagen peptides showed inverse relationships dependent on change in BMI status during the study. Conclusion. Six-month treatment with the non-selective NSAID sulindac was associated with changes in collagen-associated peptide differently by BMI status and by weight change. Our findings are most consistent with changes in post-translational hydroxylated proline modifications of collagen variation in the triple helical region. Ongoing work may provide insights on inflammation/adiposity-associated inflammation and effects on breast tissue collagen. Citation Format: Denys Rujchanarong, Peggi M. Angel, Alison Stopeck, Christina Preece, Pavani Chalasani, Patricia A. Thompson. Evidence that body mass index modifies breast tissue collagen peptide response pattern to treatment with the non-steroidal anti-inflammatory drug sulindac [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-10-05.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Tissues via the Formation of Reproducible Matrix Crystals by the Fluorescence-Assisted Spraying Method: A Quantification Approach

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging to quantitative analyses is restricted by the variability of MS intensity of the analytes in nonreproducible matrix crystals of tissues. To overcome this challenge, a fluorescence-assisted spraying method was developed for a constant matrix amount employing an MS-detectable fluorescent reagent, rhodamine 6G (R6G), which was sprayed with the matrix. To form a homogeneous matrix crystal on the tissue section, a matrix solution, 1,5-diaminonaphthalene (10 mg/mL), containing R6G (40 μg/mL) and O-dinitrobenzene (O-DNB, 10 mg/mL) was sprayed until the desired constant fluorescence intensity was achieved. Compared with that obtained via conventional cycle-number-fixed spraying [relative standard deviation (RSD) = 31.1%], the reproducibility of the relative MS intensity of the analyte [ferulic acid (FA), RSD = 3.1%] to R6G was significantly improved by the fluorescence-assisted matrix spraying. This result indicated that R6G could be employed as an index of the matrix amount and an MS normalizing standard. The proposed matrix spraying successfully quantified nifedipine (0.5-40 pmol/mm2 in the positive mode, R2 = 0.965) and FA (0.5-75 pmol/mm2 in the negative mode, R2 = 0.9972) in the kidney section of a rat. Employing the quantitative MALDI-MS imaging assay, FA, which accumulated in the kidney of the rat after 50 mg/kg was orally administered, was visually determined to be 3.5, 3.0, and 0.2 μmol/g tissue at 15, 30, and 60 min, respectively.

Insights and prospects for ion mobility-mass spectrometry in clinical chemistry

Introduction : Ion mobility-mass spectrometry is an emerging technology in the clinical setting for high throughput and high confidence molecular characterization from complex biological samples. Ion mobility spectrometry can provide isomer separations on the basis of molecular structure, the ability of which is increasing through technological developments that afford enhanced resolving power. Integrating multiple separation dimensions, such as liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) provide dramatic enhancements in the mitigation of molecular interferences for high accuracy clinical measurements. Areas covered : Multidimensional separations with LC-IM-MS provide better selectivity and sensitivity in molecular analysis. Mass spectrometry imaging of tissues to inform spatial molecular distribution is improved by complementary ion mobility analyses. Biomarker identification in surgical environments is enhanced by intraoperative biochemical analysis with mass spectrometry and holds promise for integration with ion mobility spectrometry. New prospects in high resolving power ion mobility are enhancing analysis capabilities, such as distinguishing isomeric compounds. Expert Opinion : Ion mobility-mass spectrometry holds many prospects for the field of isomer identification, molecular imaging, and intraoperative tumor margin delineation in clinical settings. These advantages are afforded while maintaining fast analysis times and subsequently high throughput. High resolving power ion mobility will enhance these advantages further, in particular for analyses requiring high confidence isobaric selectivity and detection.

Direct Tissue Mass Spectrometry Imaging by Atmospheric Pressure UV-Laser Desorption Plasma Postionization.

Matrix-assisted laser desorption ionization (MALDI) operated at atmospheric pressure has been shown to be a promising technique for mass spectrometry imaging of biological tissues at high spatial resolution. Recent studies have shown several orders of magnitude improvement in sensitivity afforded by coupling with a low-temperature plasma (LTP) for postionization. In this work we report the first results from "matrix-free" imaging using our atmospheric pressure (AP) transmission mode (TM) (MA)LDI source with LTP postionization. Direct MSI analysis of murine testis with no sample preparation after tissue sectioning enabled imaging of a range of lipid classes at pixel sizes of 25 μm. We compared results from the matrix-free methods with MALDI experiments in which the matrix was applied on top, underneath, or layered as a sandwich. The sandwich preparation was found to lead to ion yields approximately 2- or 3-fold higher than the other methods, indicating that the addition of a light absorbing matrix remains beneficial. Nonetheless, LDI methods confer a range of advantages, and the sensitivity improvements provided by postionization strategies are a promising step toward high-efficiency laser sampling under ambient conditions.

Abstract C032: Distinguishing lethal from indolent prostate cancer using N-glycan imaging mass spectrometry in a racial tissue microarray cohort

Abstract Changes in cell surface protein glycosylation are common alterations that occur with tumor progression and reflect the use of many cancer biomarkers like PSA and CA19-9. However, identification of specific glycans associated with tumor regions is still poorly defined. Our group has developed a two-dimensional glycan tissue imaging mass spectrometry approach that can be used with any clinical formalin-fixed, paraffin-embedded tumor tissue used in pathology. Based on the analysis of over one thousand prostate cancer FFPE tissue blocks and tissue microarray samples across the spectrum of disease, it was found that the presence of multi-fucosylated branched N-glycans is associated with advanced tumors with neuroendocrine and metastatic features. These glycans are not present in indolent and lower-grade adenocarcinomas. The hypothesis currently being tested is to determine whether detection of higher numbers of these fucosylated structures predicts a worse prognosis for progressive metastatic disease. Race could be a factor for increased presentation at the time of diagnosis with more advanced disease. The goal of the present study was to assess whether more advanced tumors containing multi-fucosylated N-glycans are detected more frequently in a racial cohort of 307 samples. A series of 14 prostate tumor tissue microarrays (TMA) representing African-American (n=105), Hispanic (n=101) and Caucasian (n=101) subjects was evaluated with this method. The 307 individual tumor samples represented the spectrum of tumor stage, Gleason grade, and status of disease recurrence. For select cases, the original source tumor block tissues were analyzed to confirm the TMA core results. Each TMA slide was processed for antigen retrieval and peptide N-glycosidase F digestions to release N-glycans. Samples were analyzed by MALDI-FTICR mass spectrometry, and data were visualized and analyzed by SCiLS Lab software. Tumor tissues having increased levels of fucosylation were detected in each cohort, but with an overall increase in numbers detected in the African-American cohort. Multitiered analyses are ongoing to identify glycan signatures associated with age, grade, stage, race, and recurrence. In summary, the presence and detection of a distinct panel of multi-fucosylated tissue N-glycans associated with the most lethal forms of prostate cancer can be detected in low-grade tumor samples at the time of diagnosis. This could be developed into a tissue-based prognostic biomarker panel to be applied at the time of initial diagnosis, and impact earlier treatment decisions. Citation Format: Richard R. Drake, Fred David, Cameron Miller, Melanie Jefferson, Laura Spruill, Michael Liss, Brandi Weaver, Peggi M. Angel, Robin Leach, Chanita Hughes-Halbert. Distinguishing lethal from indolent prostate cancer using N-glycan imaging mass spectrometry in a racial tissue microarray cohort [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C032.

Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissue.

The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases. We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15μm) of regions of interest and leads to reduced time requirements for data acquisition. MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation. The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.

Development and evaluation of matrix application techniques for high throughput mass spectrometry imaging of tissues in the clinic

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a sensitive label-free technique that can be used to study a wide variety of clinical phenotypes. In this context, MSI offers huge diagnostic potential by supporting decision making in the determination of personalized treatment strategies. However, improvements in throughput and robustness are still needed before it finds a place in routine application. While the field has seen tremendous improvements in the throughput of data acquisition, robust and high-throughput sample preparation methods compatible with these acquisition methods need to be developed. To address this challenge, we have developed several methods to reduce the matrix application time to less than 5 min, while maintaining sensitivity and reproducibility. Workflows incorporating these methods provide a pipeline analysis time for MSI sample preparation and acquisition of less than 30 min. The reduced time for these analyses will contribute towards the integration of MSI into routine molecular pathology for clinical diagnostics.

High-Resolution Tissue Mass Spectrometry Imaging Reveals a Refined Functional Anatomy of the Human Adult Adrenal Gland.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.

Vitamin D prevents lipid accumulation in murine muscle through regulation of PPARγ and perilipin-2 expression

Vitamin D plays an important role in regulation of skeletal muscle tone and contraction. Serum vitamin D status is linked to muscle power and force in adolescent girls, and vitamin D deficiency is associated with myopathies in children and poorer physical performance in the elderly. We previously reported that vitamin D deficiency is linked to a significant increase in muscle fatty infiltration in healthy young women, and studies in patients with neuromuscular disorders also associate muscle weakening and lipid content. In order to better understand the link between vitamin D status and skeletal muscle lipid metabolism, we compared the effect of a low (25 IU/kg) or normal (1000 IU/kg) vitamin D3 diet on muscle fat in female FVB mice maintained in a room without UVB lighting to minimize endogenous vitamin D production. Animals on low vitamin D diet displayed lower circulating 25(OH)D levels and a dramatic increase (287 ± 52% compared to normal diet, p < 0.0001) in lipid deposition in skeletal muscle accompanied by muscle fiber disorganization. Lipid droplet staining increased by 242 ± 23% (p < 0.0001) in low vitamin D diet, and lipid droplet coat protein perilipin-2 and nuclear receptor transcription factor PPARγ expression levels were increased compared to mice fed the normal vitamin D diet: average staining for PLIN2: 0.22 ± 0.08 (25 IU/kg diet) vs 0.10 ± 0.02 (1000 IU/kg). Average staining for PPARγ: 0.24 ± 0.06 (25 IU/kg diet) vs 0.07 ± 0.04 (1000 IU/kg) p < 0.0001. Tissue mass spectrometry imaging revealed major differences in muscle phospholipids profile depending on diet. In vitro, 1,25(OH)2D3 treatment of 3T3-L1 pre-adipocytes inhibited appearance of lipid droplets by 79 ± 9.3%, and caused a 80 ± 10% and 25 ± 8% (p = 0.001) reduction in PPARγ and perilipin-2 mRNA levels (by qPCR) compared to control cells. In summary, we report here the first in vivo model illustrating the important structural muscle fiber disorganization and fat accumulation inside and outside muscle fibers that accompany vitamin D deficiency. Furthermore, we show that the underlying mechanisms involve PPARγ and perilipin-2.

High resolution tissue mass spectrometry imaging: a new tool for identification of prognostic markers in adrenocortical carcinoma

High resolution tissue mass spectrometry imaging: a new tool for identification of prognostic markers in adrenocortical carcinoma

A Simple Method for Improving the Spatial Resolution in Infrared Laser Ablation Mass Spectrometry Imaging.

In mass spectrometry imaging of tissues, the size of structures that can be distinguished is determined by the spatial resolution of the imaging technique. Here, the spatial resolution of IR laser ablation is markedly improved by increasing the distance between the laser and the focusing lens. As the distance between the laser and the lens is increased from 1 to 18 m, the ablation spot size decreases from 440 to 44 μm. This way, only the collimated center of the divergent laser beam is directed on the focusing lens, which results in better focusing of the beam. Part of the laser energy is lost at longer distance, but this is compensated by focusing of the radiation to a smaller area on the sample surface. The long distance can also be achieved by a set of mirrors, between which the radiation travels before it is directed to the focusing lens and the sample. This method for improving the spatial resolution can be utilized in mass spectrometry imaging of tissues by techniques that utilize IR laser ablation, such as laser ablation electrospray ionization, laser ablation atmospheric pressure photoionization, and matrix-assisted laser desorption electrospray ionization. Graphical Abstract ᅟ.

Corneal haze phenotype in Aldh3a1-null mice: In vivo confocal microscopy and tissue imaging mass spectrometry

ALDH3A1 is a corneal crystallin that protects ocular tissues from ultraviolet radiation through catalytic and non-catalytic functions. In addition, ALDH3A1 plays a functional role in corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation. We have previously shown that Aldh3a1 knockout mice in a C57B6/129sV mixed genetic background develop lens cataracts. In the current study, we evaluated the corneal phenotype of Aldh3a1 knockout mice bred into a C57B/6J congenic background (KO). In vivo confocal microscopy examination of KO and wild-type (WT) corneas revealed KO mice to exhibit corneal haze, manifesting marked light scattering from corneal stroma. This corneal phenotype was further characterized by Imaging Mass Spectrometry (IMS) with spatial resolution that revealed a trilayer structure based on differential lipid localization. In these preliminary studies, no differences were observed in lipid profiles from KO relative to WT mice; however, changes in protein profiles of acyl-CoA binding protein (m/z 9966) and histone H4.4 (m/z 11308) were found to be increased in the corneal epithelial layer of KO mice. This is the first study to use IMS to characterize endogenous proteins and lipids in corneal tissue and to molecularly explore the corneal haze phenotype. Taken together, the current study presents the first genetic animal model of cellular-induced corneal haze due to the loss of a corneal crystallin, and strongly supports the notion that ALDH3A1 is critical to cellular transparency. Finally, IMS represents a valuable new approach to reveal molecular changes underlying corneal disease.

Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution.

We report an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) setup with a lateral resolution of 1.4 μm, a mass resolution greater than 100,000, and accuracy below ±2 p.p.m. We achieved this by coupling a focusing objective with a numerical aperture (NA) of 0.9 at 337 nm and a free working distance of 18 mm in coaxial geometry to an orbitrap mass spectrometer and optimizing the matrix application. We demonstrate improvement in image contrast, lateral resolution, and ion yield per unit area compared with a state-of-the-art commercial MSI source. We show that our setup can be used to detect metabolites, lipids, and small peptides, as well as to perform tandem MS experiments with 1.5-μm2 sampling areas. To showcase these capabilities, we identified subcellular lipid, metabolite, and peptide distributions that differentiate, for example, cilia and oral groove in Paramecium caudatum.

Secondary Ion Mass Spectrometry Imaging of Tissues, Cells, and Microbial Systems.

Secondary ion mass spectrometry (SIMS) is a technique capable of imaging tissues, single cells, and microbes revealing chemical species with sub-micrometer spatial resolution. The recently developed Fourier transform ion cyclotron resonance (FTICR) SIMS instrument provides high mass resolving power and mass accuracy, ToF-SIMS can generate chemical maps with an order of magnitude better lateral resolution than the FTICR-SIMS, and the NanoSIMS instrument offers sub-100 nm spatial resolution in chemical imaging. Many commercial ToF-SIMS instruments are also capable of depth profiling that allows three-dimensional reconstructions of cell and tissue structure.

Comprehensive bioimaging with fluorinated nanoparticles using breathable liquids.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.