Gastrointestinal cancer organoids—applications in basic and translational cancer research

Cancer is a major health problem and a leading cause of death worldwide. Early cancer detection and continuous changes in treatment strategies have improved overall patient survival. The recent development of targeted drugs offers new opportunities for personalized cancer treatment. Nevertheless, individualized treatment is accompanied by the need for biomarkers predicting the response of a patient to a certain drug. One of the most promising breakthroughs in recent years that might help to overcome this problem is the organoid technology. Organoid cultures exhibit self-renewal capacity, self-organization, and long-term proliferation, while recapitulating many aspects of their primary tissue. Generated patient-derived organoid (PDO) libraries constitute “living” biobanks, allowing the in-depth analysis of tissue function, development, tumor initiation, and cancer pathobiology. Organoids can be derived from all gastrointestinal tissues, including esophageal, gastric, liver, pancreatic, small intestinal and colorectal tissues, and cancers of these tissues. PDOs are amenable to various techniques, including sequencing analyses, drug screening, targeted therapy testing, tumor microenvironment studies, and genetic engineering capabilities. In this review, we discuss the different applications of gastrointestinal organoids in basic cancer biology and clinical translation.Subject terms: Gastrointestinal cancer, Cancer models     

Novel vacuum stable ketone‐based matrices for high spatial resolution MALDI imaging mass spectrometry

We describe the use of aromatic ketones and cinnamyl ketones that have high vacuum stability for analyzing tissue sections using matrix‐assisted laser desorption/ionization imaging mass spectrometry. Specifically, the matrix, (E)‐4‐(2,5‐dihydroxyphenyl)but‐3‐en‐2‐one (2,5‐cDHA) provides high sensitivity and high vacuum stability while producing small size crystals (1‐2 μm). A high throughput and highly reproducible sample preparation method was developed for these matrices that first involves using an organic spray solution for small matrix crystal seeding followed by spraying of the matrix in a 30% acetonitrile/70% water solution on the tissue surface to obtain a homogeneous coating of small crystals, suitable for high spatial resolution imaging.     

3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry

In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self-organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long-term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ-on-a-chip” systems. Organ-on-a-chip technology allows a broad range of “on-chip” and “off-chip” analytical techniques, whereby “on-chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on-chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.     

Immersion by rotation‐based application of the matrix for fast and reproducible sample preparations and robust results in mass spectrometry imaging

Automated matrix deposition for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is crucial for producing reproducible analyte ion signals. Here we report an innovative method employing an automated immersion apparatus, which enables a robust matrix deposition within 5 minutes and with scalable throughput by using MAPS matrix and non‐polar solvents. MSI results received from mouse heart and rat brain tissues were qualitatively similar to those from nozzle sprayed samples with respect to peak number and quality of the ion images. Overall, the immersion‐method enables a fast and careful matrix deposition and has the future potential for implementation in clinical tissue diagnostics.     

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra‐high‐performance liquid–high‐resolution tandem mass spectrometry for topical bioequivalence evaluation

Time–concentration curves for the topical anti‐viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high‐throughput ultra‐high‐performance liquid chromatography–high‐resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1–25 ng/mL for a sample volume of just 20 μL and employed cation‐exchange solid‐phase extraction in a fully automated sample treatment procedure. Short‐ and long‐term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.     

Molecular histology analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix‐assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent‐free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region‐specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed. Copyright © 2011 John Wiley & Sons, Ltd.     

High‐Throughput Proteomics Enabled by a Photocleavable Surfactant

Mass spectrometry (MS)‐based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high‐throughput proteomics. Herein, we report a high‐throughput bottom‐up proteomic method enabled by a newly developed MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS‐analysis following UV degradation. Moreover, we developed an Azo‐aided bottom‐up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all‐in‐one” MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications.     

Comparison of hydrodynamically closed two‐dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un‐pretreated urine matrices. An alternative approach, two‐dimensional (column‐coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low‐cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on‐line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5‐fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.     

Development,validation and comparison of four methods for quantifying endogenous 25OH‐D3 in human plasma

To meet the increasing clinical needs for 25‐hydroxyvitamin D3 (25OH‐D3) detection, the development of an efficient and accurate high‐performance liquid chromatography–mass spectrometry (HPLC–MS) method for plasma 25OH‐D3 quantitation is important. Since 25OH‐D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH‐D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid–liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography–tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.     

Desorption sonic spray ionization for (high) voltage-free ambient mass spectrometry

Sonic spray ionization is shown to create a supersonic cloud of charged droplets able to promote efficient desorption and ionization of drugs directly from the surfaces of commercial drug tablets at ambient conditions. Compared with desorption electrospray ionization (DESI), desorption sonic spray ionization (DeSSI) is advantageous since it uses neither heating nor high voltages at the spray capillary. DeSSI therefore provides a more friendly environment in which to perform ambient mass spectrometry (MS). DeSSI-MS is herein evaluated for the analysis of drug tablets, and found to be, in general, as sensitive as DESI-MS. The (high) voltage-free DeSSI method provides, however, cleaner mass spectra with less abundant solvent cluster ions and with enough abundant analyte signal for tandem mass spectrometry (MS/MS). These features may therefore facilitate the DeSSI-MS detection of low molar mass components or impurities, or both. The higher-velocity supersonic DeSSI spray also facilitates matrix penetration thus providing more homogenous sampling and longer lasting ion signals.     

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C₁₈ column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC₅₀ values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.     

High‐throughput salting‐out assisted liquid–liquid extraction with acetonitrile for the determination of anandamide in plasma of hemodialysis patients with liquid chromatography tandem mass spectrometry

Anandamide (AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. However, low AEA plasma levels pose challenges in terms of analytical characterization. Classical liquid‐based lipid extraction and solid‐phase extraction require complicated procedures and the drying down of relatively large volumes of solvents, making them unsuitable for high‐throughput analysis. Here a high‐throughput salting‐out assisted liquid–liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis of AEA in human plasma has been developed and validated. The seamless interface of SALLE and LC‐MS eliminated the drying‐down step, only 100 μL of plasma is required and minimal volumes of organic solvent are used. Good reproducibility, accuracy and precision were demonstrated during the method validation. The method is linear up to 10 ng/mL with a lower limit of quantitation of 0.1 ng/mL for AEA, the accuracy for AEA was from 93.3 to 96.7% and the precision was <8.57%. This new methodology was successfully applied to analysis of clinical samples from maintenance hemodialysis patients. Copyright © 2015 John Wiley & Sons, Ltd.     

Silver‐109‐based laser desorption/ionization mass spectrometry method for detection and quantification of amino acids

A new methodology applicable for both high‐resolution laser desorption/ionization mass spectrometry and mass spectrometry imaging of amino acids is presented. The matrix‐assisted laser desorption ionization‐type target containing monoisotopic cationic ¹⁰⁹Ag nanoparticles (¹⁰⁹AgNPs) was used for rapid mass spectrometry measurements of 11 amino acids of different chemical properties. Amino acids were directly tested in 100,000‐fold concentration change conditions ranging from 100 μg/mL to 1 ng/mL which equates to 50 ng to 500 fg of amino acid per measurement spot. Limit of detection values obtained suggest that presented method/target system is among the fastest and most sensitive ones in laser mass spectrometry. Mass spectrometry imaging of spots of human blood plasma spiked with amino acids showed their surface distribution allowing optimization of quantitative measurements.     

Assessment of intracellular methotrexate and methotrexate-polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry

A new ultrafast quantitative and high‐throughput mass spectrometric method using matrix‐assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid‐phase extraction of MTX and MTX‐polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within‐run and between‐run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal‐to‐noise (S/N) ratio = 3:1 in drug‐ free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX‐polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low‐dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10⁶ erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX‐polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients. Copyright © 2011 John Wiley & Sons, Ltd.     

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers

In this work, we combined a newly developed matrix coating technique – matrix coating assisted by an electric field (MCAEF) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal‐to‐noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500–37500 mass range on a time‐of‐flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100‐A9, S100‐A10, and S100‐A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase kinase 2, a fragment of cAMP‐regulated phosphoprotein 19, 3 apolipoproteins (C‐I, A‐I, and A‐II), 2 S100 proteins (A6 and A8), β‐microseminoprotein, tumor protein D52, α‐1‐acid glycoprotein 1, heat shock protein β‐1, prostate‐specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t‐test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C‐I, S100‐A6, and S100‐A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI‐MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF‐MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.     

Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids

In the last decade various analytical strategies have been established to enhance separation speed and efficiency in high performance liquid chromatography applications. Chromatographic supports based on monolithic material, small porous particles, and porous layer beads have been developed and commercialized to improve throughput and separation efficiency. This paper provides an overview of current developments in fast chromatography combined with mass spectrometry for the analysis of metabolites and proteins in clinical applications. Advances and limitations of fast chromatography for the combination with mass spectrometry are discussed. Practical aspects of, recent developments in, and the present status of high-throughput analysis of human body fluids for therapeutic drug monitoring, toxicology, clinical metabolomics, and proteomics are presented.     

A sensitive and selective HPLC–MS3 method for therapeutic drug monitoring of meropenem and its validation by comparison with HPLC–MS2 methods

Meropenem, a representative β-lactam antibiotic, is widely used to treat complicated and serious infections. Therefore, it is of great significance to monitor the plasma drug concentration for individualized antimicrobial therapy. This study first describes the development and validation of high-performance liquid chromatography–tandem mass spectrometry cubed method for monitoring meropenem in human plasma. Protein precipitation with methanol and a chromatographic analysis time of 7 min make this method simple and of high throughput. Meropenem was extracted from human plasma with recoveries >94.1%. Calibration curves were linear (R² > 0.995) in the concentration range of 0.5–50 μg/mL. Overall accuracy and precision did not exceed 8.0% as well as no significant matrix effect was observed. The novelty of this method is that the triple-stage mass spectrometry technology improves the selectivity and sensitivity. A comparison of the presented method and traditional liquid chromatography–tandem mass spectrometry method was assessed in 44 patients treated with meropenem and Passing–Bablok regression coefficients and Bland–Altman plots showed that no significant difference between the two methods. So the triple-stage mass spectrometry method developed in this study is appropriate and practical for the monitor of meropenem in the daily clinical laboratory practice.     

Screening and investigation of triplex DNA binders from Stephania tetrandra S. Moore by a combination of peak area‐fading ultra high‐performance liquid chromatography with orbitrap mass spectrometry and optical spectroscopies

The identification and screening of triplex DNA binders are important because these compounds, in many cases, are potential anticancer agents as well as promising drug candidates. Therefore, the ability to screen for these compounds in a high‐throughput mode could dramatically improve the drug screening process. A method involving a combination of 96‐well plate format and peak area‐fading ultra high‐performance liquid chromatography coupled with Orbitrap mass spectrometry was employed for screening bioactive compounds binding to the triplex DNA from the extracts of Stephania tetrandra S. Moore. Two compounds were screened out and identified as fangchinoline and tetrandrine based on the comparison of retention time and tandem mass spectrometry data with those of standards. The binding mechanisms of fangchinoline and tetrandrine at the molecular level were explored using tandem mass spectrometry, fluorescence spectroscopy, ultraviolet‐visible spectroscopy, and circular dichroism. Collision‐induced dissociation experiments showed that the complexes with fangchinoline and tetrandrine were dissociated by ligand elimination. According to these measurements, an intercalating binding is the most appropriate binding mode of these two alkaloids to the triplex DNA. The current work provides not only deep insight into alkaloid‐triplex DNA complexes but also useful guidelines for the design of efficient anticancer agents.