Imaging Mass Cytometry and Single-Cell Genomics Reveal Differential Depletion and Repletion of B-Cell Populations Following Ofatumumab Treatment in Cynomolgus Monkeys.

Ofatumumab is the first, fully human, anti-CD20 monoclonal antibody in Phase 3 development for multiple sclerosis (MS). The study focused on changes in lymphocyte subsets in blood and lymphoid tissues and on potential novel biomarkers as a result of anti-CD20 antibody action in Cynomolgus monkeys treated with human equivalent doses of subcutaneous (s.c.) ofatumumab on Days 0, 7, and 14. Axillary lymph nodes (LNs) and blood samples were collected at various time points until Day 90. Lymphocyte subsets were quantified by flow cytometry, while morphological and immune cell changes were assessed by imaging mass cytometry (IMC), immunohistochemistry (IHC), in situ hybridization (ISH), and transcriptome analyses using single-cell methodology. Ofatumumab treatment resulted in a potent and rapid reduction of B cells along with a simultaneous drop in CD20+ T cell counts. At Day 21, IHC revealed B-cell depletion in the perifollicular and interfollicular area of axillary LNs, while only the core of the germinal center was depleted of CD20+CD21+ cells. By Day 62, the perifollicular and interfollicular areas were abundantly infiltrated by CD21+ B cells and this distribution returned to the baseline cytoarchitecture by Day 90. By IMC CD20+CD3+CD8+ cells could be identified at the margin of the follicles, with a similar pattern of distribution at Day 21 and 90. Single-cell transcriptomics analysis showed that ofatumumab induced reversible changes in t-distributed stochastic neighbor embedding (t-SNE) defined B-cell subsets that may serve as biomarkers for drug action. In summary, low dose s.c. ofatumumab potently depletes both B cells and CD20+ T cells but apparently spares marginal zone (MZ) B cells in the spleen and LN. These findings add to our molecular and tissue-architectural understanding of ofatumumab treatment effects on B-cell subsets.

Analysis of small molecule antibody-drug conjugate catabolites in rat liver and tumor tissue by liquid extraction surface analysis micro-capillary liquid chromatography/tandem mass spectrometry.

RATIONALE: Antibody-drug conjugates (ADCs) are some of the most promising antibody-related therapeutics. The fate of the cytotoxic moiety of ADCs in vivo after proteolytic degradation of the antibody needs to be well understood in order to mitigate toxicity risks and design proper first in patient studies. METHODS: The feasibility of liquid extraction surface analysis micro-capillary liquid chromatography/tandem mass spectrometry (LESA-µLC/MS/MS) was tested for direct surface sampling of two possible ADC catabolites composed of synthetically modified maytansinoid (DM1) and 4-[N-maleimidomethyl]cyclohexane-1-carbonyl (MCC) from rat liver and tumor tissue. Moreover, the iMatrixSpray was incorporated to prepare calibration standards (Cs) and quality control (QC) samples by spraying analyte solution at different concentrations directly on blank tissue. RESULTS: Lys-MCC-DM1 sprayed on blank liver tissue was homogeneously distributed (12.3% variability). The assay was selective (inference ≤20%) and linear from 50.0 to 1000 ng/mL without any carry-over. Inter-run accuracy and precision were ≤2.3% and ≤25.9% meeting acceptance. Lys-MCC-DM1 was the only catabolite detected in liver and tumor tissue and was most likely responsible for the total radioactivity signal in liver tissue 72 h post-dose measured by quantitative whole body autoradiography (QWBA). CONCLUSIONS: Both analytical assays (LESA-µLC/MS/MS and QWBA) are complementary to each other and provide useful quantitative and qualitative information in spatial tissue distribution of ADCs and their related catabolites. Copyright © 2016 John Wiley & Sons, Ltd.

Reproducible Matrix Deposition for MALDI MSI Based on Open-Source Software and Hardware.

The new open-source software and hardware matrix deposition device named iMatrixSpray was optimized and specified for homogeneity, reproducibility, and sensitivity in MS imaging experiments. The results confirm the design claims, with the device delivering uniform coatings with a constant quality from experiment to experiment. The robustness in combination with the open design allows developing and sharing of matrix deposition and sample preparation protocols between labs. This tool therefore enables researchers to enter the field of MALDI MSI without previous experience in matrix coating.

Mass spectrometry imaging of biological tissue: an approach for multicenter studies.

Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies.

Evaluation of sparfloxacin distribution by mass spectrometry imaging in a phototoxicity model.

Mass spectrometry imaging (MSI) was applied to samples from mouse skin and from a human in vitro 3D skin model in order to assess its suitability in the context of photosafety evaluation. MSI proved to be a suitable method for the detection of the model compound sparfloxacin in biological tissues following systemic administration (oral gavage, 100 mg/kg) and subsequent exposure to simulated sunlight. In the human in vitro 3D skin model, a concentration-dependent increase as well as an irradiation-dependent decrease of sparfloxacin was observed. The MSI data on samples from mouse skin showed high signals of sparfloxacin 8 h after dosing. In contrast, animals irradiated with simulated sunlight showed significantly lower signals for sparfloxacin starting already at 1 h postirradiation, with no measurable intensity at the later time points (3 h and 6 h), suggesting a time- and irradiation-dependent degradation of sparfloxacin. The acquisition resolution of 100 µm proved to be adequate for the visualization of the distribution of sparfloxacin in the gross ear tissue samples, but distinct skin compartments were unable to be resolved. The label-free detection of intact sparfloxacin was only the first step in an attempt to gain a deeper understanding of the phototoxic processes. Further work is needed to identify the degradation products of sparfloxacin implicated in the observed inflammatory processes in order to better understand the origin and the mechanism of the phototoxic reaction.

iMatrixSpray: a free and open source sample preparation device for mass spectrometric imaging.

A device was built for matrix deposition in mass spectrometric imaging. This spray-type instrument requires no user interaction other than providing the spray solution and selecting the pre-defined or custom-built method. Robustness was achieved by utilizing a delta-robotics design in combination with a simple liquid system. All the information describing the systems is provided as open source and hardware and the design is therefore suitable for wide distribution and adaption by the scientific community.

Biodistribution and metabolism studies of lipid nanoparticle-formulated internally [3H]-labeled siRNA in mice.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

MSiMass list: a public database of identifications for protein MALDI MS imaging.

The clinical application of mass spectrometry imaging has developed into a sizable subdiscipline of proteomics and metabolomics because its seamless integration with pathology enables biomarkers and biomarker profiles to be determined that can aid patient and disease stratification (diagnosis, prognosis, and response to therapy). Confident identification of the discriminating peaks remains a challenge owing to the presence of nontryptic protein fragments, large mass-to-charge ratio ions that are not efficiently fragmented via tandem mass spectrometry or a high density of isobaric species. A public database of identifications has been initiated to aid the clinical development and implementation of mass spectrometry imaging. The MSiMass list database ( www.maldi-msi.org/mass ) enables users to assign identities to the peaks observed in their experiments and provides the methods by which the identifications were obtained. In contrast with existing protein databases, this list is designed as a community effort without a formal review panel. In this concept, authors can freely enter data and can comment on existing entries. In such, the database itself is an experiment on sharing knowledge, and its ability to rapidly provide quality data will be evaluated in the future.

Software tools of the Computis European project to process mass spectrometry images.

Among the needs usually expressed by teams using mass spectrometry imaging, one that often arises is that for user-friendly software able to manage huge data volumes quickly and to provide efficient assistance for the interpretation of data. To answer this need, the Computis European project developed several complementary software tools to process mass spectrometry imaging data. Data Cube Explorer provides a simple spatial and spectral exploration for matrix-assisted laser desorption/ionisation-time of flight (MALDI-ToF) and time of flight-secondary-ion mass spectrometry (ToF-SIMS) data. SpectViewer offers visualisation functions, assistance to the interpretation of data, classification functionalities, peak list extraction to interrogate biological database and image overlay, and it can process data issued from MALDI-ToF, ToF-SIMS and desorption electrospray ionisation (DESI) equipment. EasyReg2D is able to register two images, in American Standard Code for Information Interchange (ASCII) format, issued from different technologies. The collaboration between the teams was hampered by the multiplicity of equipment and data formats, so the project also developed a common data format (imzML) to facilitate the exchange of experimental data and their interpretation by the different software tools. The BioMap platform for visualisation and exploration of MALDI-ToF and DESI images was adapted to parse imzML files, enabling its access to all project partners and, more globally, to a larger community of users. Considering the huge advantages brought by the imzML standard format, a specific editor (vBrowser) for imzML files and converters from proprietary formats to imzML were developed to enable the use of the imzML format by a broad scientific community. This initiative paves the way toward the development of a large panel of software tools able to process mass spectrometry imaging datasets in the future.

imzML--a common data format for the flexible exchange and processing of mass spectrometry imaging data.

The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.

Mass spectrometry imaging for drug distribution studies.

Since its introduction mass spectrometry imaging (MSI) has proven to be a powerful tool for the localization of molecules in biological tissues. In drug discovery and development, understanding the distribution of both drug and its metabolites is of critical importance. Traditional methods suffer from a lack of spatial information (tissue extraction followed by LCMS) or lack of specificity resulting in the inability to resolve parent drug from its metabolites (whole body autoradiography). MSI is a sensitive and label-free approach for imaging drugs and metabolites in tissues. In this article we review the different MSI technologies that have been applied to the imaging of pharmaceuticals. Recent technical advances, applications and current analytical limitations are discussed.

Going forward: Increasing the accessibility of imaging mass spectrometry.

The driving force behind the high and increasing popularity of imaging mass spectrometry is its demonstrated potential for the determination of new diagnostic/prognostic biomarkers and its ability to simultaneously trace the distributions of pharmaceuticals and their metabolites in tissues without the need to develop expensive radioactively-labeled analogues. Both of these applications would benefit from standardized methods, for the development of novel MS-based molecular histology tests and governmental-approved MS-based assays for pharmaceutical development. In addition, the broader scientific community would benefit from the increased accessibility of the technique. Currently imaging MS studies are individual endeavors, utilizing the individual expertise and infrastructure of a single laboratory and their immediate collaborators. A wide array of tissue preparation, data acquisition and data analysis techniques has been developed but lacks an international collaborative structure and data sharing capabilities. Such a collaborative framework would enable methodological exchange and detailed comparisons of analytical capabilities, to explore synergies between the different methods and result in the development of robust standardized methods. Here we describe the activities of a new European imaging MS network that will explicitly compare and contrast existing methods to provide best practice guidelines for the entire healthcare research community.

High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.

Applications of MALDI-MSI to pharmaceutical research.

MALDI-MSI has been demonstrated to be a suitable technique in pharmaceutical research for providing information of the distribution of low molecular weight compounds such as drugs and their metabolites within whole-body tissue sections. Important ADME information can be determined by MALDI-MSI analysis of the distribution of drugs and metabolites in whole-body tissue sections taken from animals killed at a range of time points postdose. In this example we applied MALDI-MSI to the localization of a compound and its primary metabolite in whole-body mouse sections.

Autoradiography, MALDI-MS, and SIMS-MS imaging in pharmaceutical discovery and development.

Whole-body autoradiography ((WBA) or quantitative WBA (QWBA)), microautoradiography (MARG), matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), and secondary ion mass spectrometric imaging (SIMS-MSI) are high-resolution, molecular imaging techniques used to study the tissue distribution of radiolabeled and nonlabeled compounds in ex vivo, in situ biological samples. WBA, which is the imaging of the whole-body of lab animals, and/or their organ systems; and MARG, which provides information on the localization of radioactivity in histological preparations and at the cellular level, are used to support drug discovery and development efforts. These studies enable the conduct of human radiolabeled metabolite studies and have provided pharmaceutical scientists with a high resolution and quantitative method of accessing tissue distribution. MALDI-MSI is a mass spectrometric imaging technique capable of label-free and simultaneous determination of the identity and distribution of xenobiotics and their metabolites as well as endogenous substances in biological samples. This makes it an interesting extension to WBA and MARG, eliminating the need for radiochemistry and providing molecular specific information. SIMS-MSI offers a complementary method to MALDI-MSI for the acquisition of images with higher spatial resolution directly from biological specimens. Although traditionally used for the analysis of surface films and polymers, SIMS has been used successfully for the study of biological tissues and cell types, thus enabling the acquisition of images at submicrometer resolution with a minimum of samples preparation.

Single bead labeling method for combining confocal fluorescence on-bead screening and solution validation of tagged one-bead one-compound libraries.

Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.

Matrix-assisted laser desorption/ionization mass spectrometric imaging of complete rat sections using a triple quadrupole linear ion trap.

The fast imaging of complete rat sections by matrix-assisted laser desorption/ionization on a triple quadrupole linear ion trap is demonstrated. After administration of the pharmaceutical compound (MW=467.4 u) at 0.5 mg/kg the parent drug could be identified in full scan mode and in the enhanced product ion spectrum mode. Furthermore, the precursor ion mode could also be used to monitor the presence of the parent drug in the tissue section. In the selected reaction monitoring mode, using a laser frequency of 1000 Hz and a rastering speed of about 18 mm/s, a targeted representative image of drug distribution in a rat section could be obtained in less than 15 min.

Dynamics of Abeta turnover and deposition in different beta-amyloid precursor protein transgenic mouse models following gamma-secretase inhibition.

Human beta-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimer's disease. We have characterized the dynamics of beta-amyloid (Abeta) generation and deposition following gamma-secretase inhibition with compound LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]. Kinetic studies in preplaque mice distinguished a detergent-soluble Abeta pool in brain with rapid turnover (half-lives for Abeta40 and Abeta42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Abeta in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Abeta pool, whereas plasma Abeta turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for gamma-secretase degradation were estimated as 0.4 and 0.1 h for C99 and C83, respectively. Three different APP transgenic lines responded very similarly to gamma-secretase inhibition regardless of the familial Alzheimer's disease mutations in APP. Amyloid deposition started with Abeta42, whereas Abeta38 and Abeta40 continued to turn over. Chronic gamma-secretase inhibition lowered amyloid plaque formation to a different degree in different brain regions of the same mice. The extent was inversely related to the initial amyloid load in the region analyzed. No evidence for plaque removal below baseline was obtained. gamma-Secretase inhibition led to a redistribution of intracellular Abeta and an elevation of CTFs in neuronal fibers. In CSF, Abeta showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Abeta in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Abeta-lowering therapeutics.