Mass Spectrometry-Based Proteomics of Poly(methylmethacrylate)-Embedded Bone.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely employed technique in proteomics research for studying the proteome biology of various clinical samples. Hard tissues, such as bone and teeth, are routinely preserved using synthetic poly(methyl methacrylate) (PMMA) embedding resins that enable histological, immunohistochemical, and morphological examination. However, the suitability of PMMA-embedded hard tissues for large-scale proteomic analysis remained unexplored. This study is the first to report on the feasibility of PMMA-embedded bone samples for LC-MS/MS analysis. Conventional workflows yielded merely limited coverage of the bone proteome. Using advanced strategies of prefractionation by high-pH reversed-phase liquid chromatography in combination with isobaric tandem mass tag labeling resulted in proteome coverage exceeding 1000 protein identifications. The quantitative comparison with cryopreserved samples revealed that each sample preparation workflow had a distinct impact on the proteomic profile. However, workflow replicates exhibited a high reproducibility for PMMA-embedded samples. Our findings further demonstrate that decalcification prior to protein extraction, along with the analysis of solubilization fractions, is not preferred for PMMA-embedded bone. The biological applicability of the proposed workflow was demonstrated using samples of human PMMA-embedded alveolar bone and the iliac crest, which revealed anatomical site-specific proteomic profiles. Overall, these results establish a crucial foundation for large-scale proteomics studies contributing to our knowledge of bone biology.

Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications.

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Removal of optimal cutting temperature (O.C.T.) compound from embedded tissue for MALDI imaging of lipids.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a common molecular imaging modality used to characterise the abundance and spatial distribution of lipids in situ. There are several technical challenges predominantly involving sample pre-treatment and preparation which have complicated the analysis of clinical tissues by MALDI-MSI. Firstly, the common embedding of samples in optimal cutting temperature (O.C.T.), which contains high concentrations of polyethylene glycol (PEG) polymers, causes analyte signal suppression during mass spectrometry (MS) by competing for available ions during ionisation. This suppressive effect has constrained the application of MALDI-MSI for the molecular mapping of clinical tissues. Secondly, the complexity of the mass spectra is obtained by the formation of multiple adduct ions. The process of analyte ion formation during MALDI can generate multiple m/z peaks from a single lipid species due to the presence of alkali salts in tissues, resulting in the suppression of protonated adduct formation and the generation of multiple near isobaric ions which produce overlapping spatial distributions. Presented is a method to simultaneously remove O.C.T. and endogenous salts. This approach was applied to lipid imaging in order to prevent analyte suppression, simplify data interpretation, and improve sensitivity by promoting lipid protonation and reducing the formation of alkali adducts.

Universal Sample Preparation Unlocking Multimodal Molecular Tissue Imaging.

A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.

A Novel HPLC-Based Method to Investigate on RNA after Fixation.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Development of a Robust Ultrasonic-Based Sample Treatment To Unravel the Proteome of OCT-Embedded Solid Tumor Biopsies.

An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.

Three-dimensional visualization of extracellular matrix networks during murine development.

The extracellular matrix (ECM) plays a crucial role in embryogenesis, serving both as a substrate to which cells attach and as an active regulator of cell behavior. However, little is known about the spatiotemporal expression patterns and 3D structure of ECM proteins during embryonic development. The lack of suitable methods to visualize the embryonic ECM is largely responsible for this gap, posing a major technical challenge for biologists and tissue engineers. Here, we describe a method of viewing the 3D organization of the ECM using a polyacrylamide-based hydrogel to provide a 3D framework within developing murine embryos. After removal of soluble proteins using sodium dodecyl sulfate, confocal microscopy was used to visualize the 3D distribution of independent ECM networks in multiple developing tissues, including the forelimb, eye, and spinal cord. Comparative analysis of E12.5 and E14.5 autopods revealed proteoglycan-rich fibrils maintain connections between the epidermis and the underlying tendon and cartilage, indicating a role for the ECM during musculoskeletal assembly and demonstrating that our method can be a powerful tool for defining the spatiotemporal distribution of the ECM during embryogenesis.

Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ-stimulated mesenchymal stromal cells that predict immunosuppression.

BACKGROUND: Although a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages. METHODS: We profiled the single-cell morphological response of MSCs from different donors and passages to the functionally relevant inflammatory cytokine interferon (IFN)-γ. We used the machine learning approach visual stochastic neighbor embedding (viSNE) to identify distinct morphological subpopulations that could predict suppression of activated CD4+ and CD8+ T cells in a multiplexed quantitative assay. RESULTS: Multiple IFN-γ-stimulated subpopulations significantly correlated with the ability of MSCs to inhibit CD4+ and CD8+ T-cell activation and served as effective CQAs to predict the immunosuppressive capacity of additional manufactured MSC lots. We further characterized the emergence of morphological heterogeneity following IFN-γ stimulation, which provides a strategy for identifying functional subpopulations for future single-cell characterization and enrichment techniques. DISCUSSION: This work provides a generalizable analytical platform for assessing functional heterogeneity based on single-cell morphological responses that could be used to identify novel CQAs and inform cell manufacturing decisions.

Development of a method to preliminarily embed tissue samples using low melting temperature fish gelatin before sectioning: A technical note.

Embedding of tissue samples that maintains a desired orientation is critical for preparing sections suitable for diagnosis and study objectives. Methods to prepare tissue sections include: (i) paraffin embedding or snap-freezing followed by microtome or cryostat sectioning; and (ii) agarose embedding followed by cutting on a vibrating microslicer. Although these methods are useful for routine laboratory work, preparation of small and fragile tissues such as mouse organs, small human biopsy samples, and cultured floating spheres is difficult and requires special skills. In particular, tissue specimen orientation can be lost during embedding in molds and subsequent sectioning. Here, we developed a method using low melting temperature (LM) gelatin either alone or mixed with agarose to preliminarily embed collected tissues that are either prefixed or unfixed, followed by conventional fixation, paraffin embedding, freezing, and sectioning. The advantage of the method is that the LM gelatin and its mixture with agarose can be handled at room temperature but quickly hardens at 4°C, which allows embedding, trimming, and arranging of small and fragile tissues in a desired orientation and are compatible with traditional stainings. Thus, this method can have various laboratory applications and can be modified according to the needs of each laboratory.

From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References.

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots.

Recently, a number of diverse correlative light and electron microscopy (CLEM) protocols have been developed for several model organisms. However, these CLEM methods have largely bypassed plant cell research, with most protocols having little application to plants. Using autophagosome identification as a biological background, we propose and compare two CLEM protocols that can be performed in most plant research laboratories, providing a good compromise that preserves fluorescent signals as well as ultrastructural features. These protocols are based on either the adaptation of a high pressure fixation/GMA acrylic resin embedding method, or on the Tokuyasu approach. Both protocols suitably preserved GFP fluorescence while allowing the observation of cell ultrastructure in plants. Finally, the advantages and disadvantages of these protocols are discussed in the context of multiscale imaging of plant cells.

Precast Gelatin-Based Molds for Tissue Embedding Compatible with Mass Spectrometry Imaging.

Preparation of tissue for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) generally involves embedding the tissue followed by freezing and cryosectioning, usually between 5 and 25 µm thick, depending on the tissue type and the analyte(s) of interest. The brain is approximately 60% fat; it therefore lacks rigidity and poses structural preservation challenges during sample preparation. Histological sample preparation procedures are generally transferable to MALDI-MSI; however, there are various limitations. Optimal cutting temperature compound (OCT) is commonly used to embed and mount fixed tissue onto the chuck inside the cryostat during cryosectioning. However, OCT contains potential interferences that are detrimental to MALDI-MSI, while fixation is undesirable for the analysis of some analytes either due to extraction or chemical modification (i.e., polar metabolites). Therefore, a method for both fixed and fresh tissue compatible with MALDI-MSI and histology is desirable to increase the breadth of analyte(s), maintain the topographies of the brain, and provide rigidity to the fragile tissue while eliminating background interference. The method we introduce uses precast gelatin-based molds in which a whole mouse brain is embedded, flash frozen, and cryosectioned in preparation for mass spectrometry imaging (MSI).

Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation.

BACKGROUND: The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. METHODS: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. RESULTS: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. CONCLUSIONS: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses.

Significance of prostate cancer missed on needle biopsy tools for retrieving missed cancer.

BACKGROUND: Prostate needle biopsy (PNB) is required for the diagnosis of prostate cancer (PCa), but little is known about the frequency and clinical implication of false-negative results. OBJECTIVE: To investigate the incidence and clinical impact of minute PCa missed on routine haematoxylin and eosin (H&E) slides, but retrieved by α-methylacyl-CoA-racemase (AMACR) immunohistochemistry. METHODS: AMACR immunohistochemistry was used to detect PCa missed on H&E slides in a series of consecutive 1,672 PNB including 1,003 patients without evidence of PCa, and 669 patients with PCa meeting pathological criteria for active surveillance (PCAS) under current clinical investigation, including Gleason score ≤7 (3 + 4), <33% of biopsies involved by cancer, <50% of any core involved by cancer. Using improved multicore (pre-) embedding techniques a single AMACR immunostain/patient was sufficient to detect missed lesions. RESULTS: In patients without histological evidence of PCa, AMACR immunohistochemistry retrieved minute PCa in 33 of 1,003 patients (3.29%) and atypical small acinar proliferations (ASAP) in 17 of 1,003 patients (1.69%). Among 116 of 669 (17.34%) PCa patients meeting PCAS, detection of additional core(s) involved by cancer was found responsible for disease reclassification in 63 of 116 of patients (54.31%). Limitations include the single-institutional design of the study. CONCLUSIONS: PCa missed on routine H&E histology was retrieved by AMACR in 8.91% of PNB, including 17.34% of PCa patients meeting PCAS. 54.31% of them have finally lost their eligibility for active surveillance after detecting additional cores involved by cancer. Underdiagnosis of limited adenocarcinoma on PNB is a matter of concern, but can be prevented by a single AMACR immunostain/patient if improved multicore (pre-) embedding techniques are used.

Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy.

Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

RATIONALE: Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. OBJECTIVES: Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. METHODS: Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. RESULTS: Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). CONCLUSIONS: ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.

3D structured illumination microscopy of mammalian embryos and spermatozoa.

BACKGROUND: Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass. RESULTS: Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa. CONCLUSIONS: Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.

Staining and embedding the whole mouse brain for electron microscopy.

The development of methods for imaging large contiguous volumes with the electron microscope could allow the complete mapping of a whole mouse brain at the single-axon level. We developed a method based on prolonged immersion that enables staining and embedding of the entire mouse brain with uniform myelin staining and a moderate preservation of the tissue's ultrastructure. We tested the ability to follow myelinated axons using serial block-face electron microscopy.