Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 µm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

Role of MALDI-MSI in combination with 3D tissue models for early stage efficacy and safety testing of drugs and toxicants.

Introduction: Three-dimensional (3D) cell cultures have become increasingly important materials to investigate biological processes and drug efficacy and toxicity. The ability of 3D cultures to mimic the physiology of primary tissues and organs in the human body enables further insight into cellular behavior and is hence highly desirable in early-stage drug development. Analyzing the spatial distribution of drug compounds and endogenous molecules provides an insight into the efficacy of a drug whilst simultaneously giving information on biological responses. Areas Covered: In this review we will examine the main 3D cell culture systems employed and applications, which describe their integration with mass spectrometry imaging (MSI). Expert Opinion: MSI is a powerful technique that can map a vast range of molecules simultaneously in tissues without the addition of labels that can provide insights into the efficacy and safety of a new drug. The combination of MSI and 3D cell cultures has emerged as a promising tool in early-stage drug analysis. However, the most common administration route for pharmaceutical drugs is via oral delivery. The use of MSI in combination with models of the GI tract is an area that has been little explored to date, the reasons for this are discussed.

Quantification of proteins in whole blood, plasma and DBS, with element-labelled antibody detection by ICP-MS.

Over recent years, quantification of multiple proteins in body fluids has become increasingly prominent, which is beneficial to a number of scientific fields, not least biomedical. Several techniques have been developed based on conventional ELISA; one of these techniques is analysis of proteins labelled with element-tagged antibodies by ICP-MS in serum, allowing quantification of multiple targets within a single sample. This research aimed to quantify albumin and immunoglobulin G (IgG) levels in plasma, whole blood and dried blood spots using NANOGOLD® and Europium labelled antibodies analysed by ICP-MS. Before the proteins were quantified simultaneously, albumin and IgG concentrations were measured separately and compared to protein levels obtained by ELISA. It was found that protein concentrations for both albumin and IgG obtained with element-labelled antibody detection correspond to those determined by ELISA. Furthermore, albumin and IgG levels measured simultaneously by ICP-MS correspond to concentrations found when the proteins were analysed separately by ICP-MS. Finally, development of this method has provided a positive indication that it can be extended to quantification of additional proteins, which could be related to a disease or as a minimum provide additional information for a protein profile of an individual.

Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDI-MSI.

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

The metabolism of 4-bromoaniline in the bile-cannulated rat: application of ICPMS ((79/81)Br), HPLC-ICPMS & HPLC-oaTOFMS.

1. An excretion balance study was performed following i.p. administration of 4-bromoaniline (50 mg kg(-1)) to bile-cannulated rats, using bromine-detected ((79/81)Br) ICPMS for quantification. Approximately 90% of the dose was recovered in urine (68.9 ± 3.6%) and bile (21.4 ± 1.4%) by 48 h post-administration. 2. HPLC-ICPMS ((79/81)Br) was used to selectively detect and profile the major urinary and biliary-excreted metabolites and determined that the 0-12 h urine contained at least 21 brominated metabolites with 19 bromine-containing peaks observed in the 6-12 h bile samples. 3. The urinary and biliary metabolites were subsequently profiled using HPLC-oaTOFMS. By exploiting the distinctive bromine isotope pattern ca. 60 brominated metabolites were detected in the urine in negative electrospray ionisation (ESI) mode while bile contained ca. 21. 4. While a large number of bromine-containing metabolites were detected, the profiles were dominated by a few major components with the bulk of the 4-bromoaniline-related material in urine accounted for by 4-bromoanaline O-sulfate (∼75% of the total by ICPMS, 84% by TOFMS). In bile a hydroxylated N-acetyl compound was the major metabolite detected, forming some ∼65% of the 4-bromoaniline-related material by ICPMS (37% by TOFMS).

Phosphorus and sulfur metabonomic profiling of tissue and plasma obtained from tumour-bearing mice using ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry.

RATIONALE: Metabonomic studies use complex biological samples (blood plasma/serum, tissues, etc.) that when analysed with high-performance liquid chromatography/mass spectrometry (HPLC/MS) or nuclear magnetic resonance (NMR) generate profiles that may contain many thousands of features. These profiles can be difficult to interpret with the majority of the features contributing little to the study. As such there is an argument for the development of techniques that can simplify the problem by targeting particular classes of compounds. METHODS: In this study ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry (UPLC/ICP-MS) was used to profile tumour tissue and plasma samples for phosphorus- and sulfur-containing metabolites. These samples were xenograft tumours (derived from breast, lung and colon cell lines) and plasma obtained from nude mice. Plasma was also obtained from non-tumour-bearing mice as a control. Due to isobaric interferences this method took advantage of the dynamic reaction cell within the ICP-MS system to react the phosphorus and sulfur ions with oxygen. The PO+ and SO+ ions were then monitored free of interferences. The total phosphorus and sulfur within each sample was also recorded using flow injection ICP-MS. A robust quality control system based on pooled sample replicate analysis was used throughout the study. RESULTS: Determination of the total phosphorus and sulfur content of each sample was sufficient in itself for statistical differentiation between the majority of the cell lines analysed. Subsequent reversed-phase chromatographic profiling of the organic tumour and plasma extracts revealed the presence of a number of well-retained phosphorus-containing compounds that showed tumour-specific profiles. Reversed-phase profiling was not suitable for the sulfur-containing compounds which eluted with the solvent front. CONCLUSIONS: This study has shown the potential use of UPLC/ICP-MS to differentiate between tumour cell lines, using both plasma and tumour tissue samples, based solely on metabolites that contain phosphorus or sulfur. Whilst further work is required to identify these compounds this methodology shows the ability of the described methods to provide targets for future biomarker discovery studies. Copyright © 2013 John Wiley & Sons, Ltd.

MALDI and Trace Metal Analysis in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains one of the most prevalent causes of blindness throughout the world. Key to prevention of AMD is furthering the understanding of its pathology. In recent years, both the proteins within the innate immune system and essential and non-essential metals have been implicated in the pathology of AMD. Herein, a multidisciplinary and multimodal methodology has been taken to further our understanding of the role of the innate immune proteins and the essential metals within mouse ocular tissue.

The Adaptation of the QV600 LLI Milli-Fluidics System to House Ex Vivo Gastrointestinal Tissue Suitable for Drug Absorption and Permeation Studies, Utilizing MALDI MSI and LC-MS/MS.

Careful formulation of pharmaceuticals for oral delivery is essential to ensure that the optimal amount of the active ingredient reaches its intended site of action. This chapter demonstrates how mass spectrometry can be used in conjunction with ex vivo tissue and an adapted milli-fluidics system to carry out a drug absorption study. MALDI MSI is used to visualize the drug within the small intestine tissue from the absorption experimentation. LC-MS/MS is used to complete a mass balance of the experiment and quantify the amount of drug that has permeated through the tissue.

University student-led public engagement event: increasing audience diversity and impact in a non-science space.

There is a wealth of innovation in microbiology outreach events globally, including in the setting where the public engagement is hosted. Previous data indicate an underrepresentation of marginalized ethnic groups attending UK science-based public engagement events. This project engaged our student cohort, encompassing a diverse range of ethnic groups, to create an integrated art and science event within an existing series of adult education evenings. The study's objectives were to increase the proportion of visitors from marginalized ethnic groups and to gain a greater understanding of the impact of the event on the visitors' reported science capital. The participants' demographics, links to our students and University, and detailed impact on participants' science capital of the event were determined through analysis of exit questionnaires. There was an increase in the proportion of marginalized ethnic group visitors compared to similar previous events. A higher proportion of visitors from marginalized ethnic groups had links with our students and University compared to white/white British visitors. Elements of the exit questionnaire were mapped to the science capital framework and participants' science capital was determined. Both ethnically marginalized participants and white/white British visitors showed an increase in science capital, specifically dimensions of science-related social capital and science-related cultural capital, after the event. In conclusion, our study suggests that a student-led blended art and science public engagement can increase the ethnic diversity of those attending and can contribute towards creating more inclusive public engagement events.

Imaging of Light-Enhanced Extracellular Vesicle-Mediated Delivery of Oxaliplatin to Colorectal Cancer Cells via Laser Ablation, Inductively Coupled Plasma Mass Spectrometry.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as 'C5') photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

Adaptation of the Kirkstall QV600 LLI Microfluidics System for the Study of Gastrointestinal Absorption by Mass Spectrometry Imaging and LC-MS/MS.

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature.

Multiomic Mass Spectrometry Imaging to Advance Future Pathological Understanding of Ocular Disease.

Determining the locations of proteins within the eye thought to be involved in ocular pathogenesis is important to determine how best to target them for therapeutic benefits. However, immunohistochemistry is limited by the availability and specificity of antibodies. Additionally, the perceived role of both essential and non-essential metals within ocular tissue has been at the forefront of age-related macular degeneration (AMD) pathology for decades, yet even key metals such as copper and zinc have yet to have their roles deconvoluted. Here, mass spectrometry imaging (MSI) is employed to identify and spatially characterize both proteomic and metallomic species within ocular tissue to advance the application of a multiomic imaging methodology for the investigation of ocular diseases.

Multi-Modal Mass Spectrometric Imaging of Uveal Melanoma.

Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI), was used to obtain images of lipids and metabolite distribution in formalin fixed and embedded in paraffin (FFPE) whole eye sections containing primary uveal melanomas (UM). Using this technique, it was possible to obtain images of lysophosphatidylcholine (LPC) type lipid distribution that highlighted the tumour regions. Laser ablation inductively coupled plasma mass spectrometry images (LA-ICP-MS) performed on UM sections showed increases in copper within the tumour periphery and intratumoural zinc in tissue from patients with poor prognosis. These preliminary data indicate that multi-modal MSI has the potential to provide insights into the role of trace metals and cancer metastasis.

Nights at the museum: integrated arts and microbiology public engagement events enhance understanding of science whilst increasing community diversity and inclusion.

This study uses integrated art and science events to explore a blended approach in improving public understanding of current scientific topics and widening participation within the local community. The events were a Halloween-inspired microbiology-themed series of interactive exhibitions hosted within a national museum as part of an existing series of adult education evenings. A representative sample of 102 mixed methods exit questionnaires, based on determining (i) audience diversity and (ii) understanding of scientific topics, were analysed by qualitative and quantitative approaches, and a post-attendance focus group was carried out to determine longer term impact of the event. Participants were grouped as 'Science', 'Arts', 'Both' or 'Neither', according to their past experience and engagement. These events welcomed more participants from the Arts and Neither subsections hence engaging a group of people who would not usually visit science public engagement events or comparative events hosted in traditional academic settings, highlighting the importance of venue choice in reaching new audiences and widening participation. An increase in perceived understanding of science was observed by all groups of participants with reported enjoyment focused around the science talks, presentations and blended art-science activities. A putative impact in science capital is observed with participants reporting an increased likelihood of attending science events in the future. Furthermore, increased discussion and awareness of science in society is evidenced by participants. Blended art and microbiology exhibitions enhance the accessibly of science public engagement events and is likely to increase science capital; the impact of this on cognitive polyphasia is also discussed.

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging.

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade.

Development and validation of a NANOGold immunoassay for the detection of vascular endothelial growth factor (VEGF) in human serum using inductively coupled plasma mass spectrometry.

This work aimed to develop and validate a NANOGold based assay, quantified using inductively coupled plasma mass spectrometry (ICP-MS), for the detection of human vascular endothelial growth factor (hVEGF) in serum. The initial assay range based on calibration standards was 62.5-2000 pg/mL with a detection limit of approximately 30 pg/mL. After validation using spiked validation controls, a quantification range between 175 and 1928 pg/mL was obtained. The inter-assay precision was between 2.3 and 18.9% with accuracy between -8.8 and -3.1%. Additional performance parameters, including dilutional linearity, matrix specificity and time-factored drift, were within +/-20%, as defined by the validation acceptance criteria for the validation of macromolecule immunoassays used within our clinical environment. Serum samples from healthy donors were analysed to determine the endogenous levels of VEGF present; these ranged from 164 to 580 pg/mL with a mean of 273 pg/mL. The intra- and inter-assay precision obtained from the healthy donor samples were 1.3-10.7% and 4.2-17.5%, respectively. This demonstration of a validated immunoassay opens further possibilities, utilising the simultaneous detection capabilities of ICP-MS for the detection of multiple analytes in a single validated immunoassay, for routine use within a clinical environment.

Profiling biological samples using ultra performance liquid chromatography-inductively coupled plasma-mass spectrometry (UPLC-ICP-MS) for the determination of phosphorus and sulfur-containing metabolites.

In this preliminary study UPLC-ICP-MS has been utilized to profile a range of different bio-fluids and tissue extracts for sulfur and phosphorus-containing metabolites. Particular attention has been given to the livers, plasma and urine from lean and obese Zucker rats, with a view to differentiating between them based solely on their respective sulfur or phosphorus profiles and/or their total sulfur and phosphorus content. In addition, bile and tumour extracts have been analysed to observe the nature of their profiles. To the best of our knowledge this is the first time ICP-MS has been used in a non-targeted metabonomic study. Results have shown lower limits of quantification for sulfur and phosphorus methods of 0.25 and 0.15 ng on column with CVs of 14.7% and 10.9% respectively. Total phosphorus analysis of the Zucker rat aqueous liver extracts, plasma and urine has shown the pattern of phosphorus concentrations to be statistically significantly different in the lean and obese Zucker rats. Chromatographic separation of the Zucker rat organic liver extracts and plasma allowed further differentiation between the lean and obese rats using their phosphorus profiles alone. In conclusion, this preliminary study has shown the potential of UPLC-ICP-MS to quantitatively discriminate between different species biofluids, fluids and tissues based solely on their phosphorus or sulfur concentrations and/or metabolomes.

Convergent evolution of cucurbitacin feeding in spatially isolated rootworm taxa (Coleoptera: Chrysomelidae; Galerucinae, Luperini).

Historically, chemical ecologists assumed that cucurbitacin feeding and sequestration in rootworm leaf beetles is a remnant of an ancient association between the Luperini (Coleoptera: Chrysomelidae; Galerucinae) and Cucurbitaceae (ancestral host hypothesis). Under this premise, rootworms that do not develop on cucurbits but undergo pharmacophagous forays for cucurbitacins are thought to do so to supplement novel host diets that lack these bitter compounds. The ancestral host hypothesis is supported from studies of pyrrolizidine alkaloid pharmacophagy in Lepidoptera but has not been subjected to phylogenetic analysis within the Luperini. New evidence that this feeding behavior is better correlated with an adult affinity for pollen than with larval host offers the possibility that Old and New World rootworm species with an affinity for cucurbitacins converged on this behavior through apomorphic taste receptor modifications (loose receptor hypothesis). Here we test the monophyly of cucurbitacin feeding within the Luperini by using nuclear and mitochondrial sequence data to infer phylogenetic relationships among 49 taxa representing tribes of the Galerucinae and subtribes of the Luperini. The resulting phylogenetic hypothesis is mostly concordant with existing tribal and subtribal delineations within the Subfamily Galerucinae sensu stricto (Galerucinae not including the flea beetles). The establishment of ancestry among the subtribes of the Luperini refutes the monophyly of cucurbitacin feeding and cucurbit specialization, with the New World Diabroticina being paraphyletic to the Old World Aulacophorina and cosmopolitan Luperina. These data unambiguously support the convergent evolution of cucurbitacin feeding in rootworms and are inconsistent with the ancestral host hypothesis.

High-performance liquid chromatography/inductively coupled plasma mass spectrometry with iodine-specific detection for profiling the metabolites produced in the earthworm Eisenia veneta by exposure to 2-fluoro-4-iodoaniline.

High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) provided a rapid and specific means for profiling the iodine-containing metabolites produced by the earthworm Eisenia veneta following exposure to 2-fluoro-4-iodoaniline. Profiles were obtained, using gradient reversed-phase HPLC, from extracts of whole earthworms and from coleomic fluid with as little as 25 ng/peak of iodine detected. The use of ICPMS in this way provides a convenient means of determining the metabolic fate of iodinated compounds without the need for radiolabelled compounds.

Tissue-specific, light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana.

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.