Volatile organic compound analysis of malignant pleural mesothelioma chorioallantoic membrane xenografts.

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM is often diagnosed late, at a point where limited treatment options are available, but early intervention could improve the chances of successful treatment for MPM patients. Biomarkers to detect MPM in at-risk individuals are needed to implement early diagnosis technologies. Volatile organic compounds (VOCs) have previously shown diagnostic potential as biomarkers when analysed in MPM patient breath. In this study, chorioallantoic membrane (CAM) xenografts of MPM cell lines were used as models of MPM tumour development for VOC biomarker discovery with the aim of generating targets for investigation in breath, biopsies or other complex matrices. VOC headspace analysis of biphasic or epithelioid MPM CAM xenografts was performed using solid-phase microextraction and gas chromatography-mass spectrometry. We successfully demonstrated the capture, analysis and separation of VOC signatures from CAM xenografts and controls. A panel of VOCs was identified that showed discrimination between MPM xenografts generated from biphasic and epithelioid cells and CAM controls. This is the first application of the CAM xenograft model for the discovery of VOC biomarkers associated with MPM histological subtypes. These findings support the potential utility of non-invasive VOC profiling from breath or headspace analysis of tissues for detection and monitoring of MPM.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Cellular Responses to Extracellular Vesicles as Potential Markers of Colorectal Cancer Progression.

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Elemental Mapping of Human Malignant Mesothelioma Tissue Samples Using High-Speed LA-ICP-TOFMS Imaging.

This is the first report of the use of laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) to analyze human malignant pleural mesothelioma (MPM) samples at the cellular level. MPM is an aggressive, incurable cancer associated with asbestos exposure, with a long latency and poor overall survival. Following careful optimization of the laser fluence, the simultaneous ablation of soft biological tissue and hard mineral fibers was possible, allowing the spatial detection of elements such as Si, Mg, Ca, and Fe, which are also present in the glass substrate. A low-dispersion LA setup was employed, which provided the high spatial resolution necessary to identify the asbestos fibers and fiber fragments in the tissue and to characterize the metallome at the cellular level (a pixel size of 2 µm), with a high speed (at 250 Hz). The multielement LA-ICP-TOFMS imaging approach enabled (i) the detection of asbestos fibers/mineral impurities within the MPM tissue samples of patients, (ii) the visualization of the tissue structure with the endogenous elemental pattern at high spatial resolution, and (iii) obtaining insights into the metallome of MPM patients with different pathologies in a single analysis run. Asbestos and other mineral fibers were detected in the lung and pleura tissue of MPM patients, respectively, based on their multielement pattern (Si, Mg, Ca, Fe, and Sr). Interestingly, strontium was detected in asbestos fibers, suggesting a link between this potential toxic element and MPM pathogenesis. Furthermore, monitoring the metallome around the talc deposit regions (characterized by elevated levels of Al, Mg, and Si) revealed significant tissue damage and inflammation caused by talc pleurodesis. LA-ICP-TOFMS results correlated to Perls' Prussian blue and histological staining of the corresponding serial sections. Ultimately, the ultra-high-speed and high-spatial-resolution capabilities of this novel LA-ICP-TOFMS setup may become an important clinical tool for simultaneous asbestos detection, metallome monitoring, and biomarker identification.

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.

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.

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.

Laser ablation inductively coupled plasma mass spectrometry as a novel clinical imaging tool to detect asbestos fibres in malignant mesothelioma.

RATIONALE: Malignant pleural mesothelioma is an extremely aggressive and incurable malignancy associated with prior exposure to asbestos fibres. Difficulties remain in relation to early diagnosis, notably due to impeded identification of asbestos in lung tissue. This study describes a novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging approach to identify asbestos within mesothelioma models with clinical significance. METHODS: Human mesothelioma cells were exposed to different types of asbestos fibres and prepared on plastic slides for LA-ICP-MS analysis. No further sample preparation was required prior to analysis, which was performed using an NWR Image 266 nm laser ablation system coupled to an Element XR sector-field ICP mass spectrometer, with a lateral resolution of 2 µm. Data was processed using LA-ICP-MS ImageTool v1.7 with the final graphic production made using DPlot software. RESULTS: Four different mineral fibres were successfully identified within the mesothelioma samples based on some of the most abundant elements that make up these fibres (Si, Mg and Fe). Using LA-ICP-MS as an imaging tool provided information on the spatial distribution of the fibres at cellular level, which is essential in asbestos detection within tissue samples. Based on the metal counts generated by the different types of asbestos, different fibres can be identified based on shape, size, and elemental composition. Detection of Ca was attempted but requires further optimisation. CONCLUSIONS: Detection of asbestos fibres in lung tissues is very useful, if not necessary, to complete the pathological dt9iagnosis of asbestos-related malignancies in the medicolegal field. For the first time, this study demonstrates the successful application of LA-ICP-MS imaging to identify asbestos fibres and other mineral fibres within mesothelioma samples. Ultimately, high-resolution, fast-speed LA-ICP-MS analysis has the potential to be integrated into clinical workflow to aid earlier detection and stratification of mesothelioma patient samples.

Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model.

Three-dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two-dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof-of-principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in-software t test and mixed-effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof-of-principle study shows for the first time that chemotherapy-induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing.

Discrimination of papillary thyroid cancer from non-cancerous thyroid tissue based on lipid profiling by mass spectrometry imaging.

INTRODUCTION: The distinction of papillary thyroid carcinomas from benign thyroid lesions has important implication for clinical man-agement. Classification based on histopathological features can be supported by molecular biomarkers, including lipidomic signatures, identified with the use of high-throughput mass spectrometry techniques. Formalin fixation is a standard procedure for stabilization and preservation of tissue samples, therefore this type of samples constitute highly valuable source of clinical material for retrospective molecular studies. In this study we used mass spectrometry imaging to detect lipids discriminating papillary cancer from not cancerous thyroid directly in formalin-fixed tissue sections. MATERIAL AND METHODS: For this purpose imaging and profiling of lipids present in non-malignant and cancerous thyroid tissue specimens were conducted. High resolution MALDI-Q-Ion Mobility-TOF-MS technique was used for lipidomic analysis of formalin fixed thyroid tissue samples. Lipids were identified by the comparison of the exact molecular masses and fragmentation pathways of the protonated molecule ions, recorded during the MS/MS experiments, with LIPID MAPS database. RESULTS: Several phosphatidylcholines (32:0, 32:1, 34:1 and 36:3), sphingomyelins (34:1 and 36:1) and phosphatidic acids (36:2 and 36:3) were detected and their abundances were significantly higher in cancerous tissue compared to non-cancerous tissue. The same lipid species were detected in formalin-fixed as in fresh-frozen tissue, but [M + Na]+ ions were the most abundant in formalin fixed whereas [M + K]+ ions were predominant in fresh tissue. CONCLUSIONS: Our results prove the viability of MALDI-MSI for analysis of lipid distribution directly in formalin-fixed tissue, and the potential for their use in the classification of thyroid diseases.

Hierarchical Cluster Analysis to Aid Diagnostic Image Data Visualization of MS and Other Medical Imaging Modalities.

Perceiving abnormal regions in the images of different medical modalities plays a crucial role in diagnosis and subsequent treatment planning. In medical images to visually perceive abnormalities' extent and boundaries requires substantial experience. Consequently, manually drawn region of interest (ROI) to outline boundaries of abnormalities suffers from limitations of human perception leading to inter-observer variability. As an alternative to human drawn ROI, it is proposed the use of a computer-based segmentation algorithm to segment digital medical image data.Hierarchical Clustering-based Segmentation (HCS) process is a generic unsupervised segmentation process that can be used to segment dissimilar regions in digital images. HCS process generates a hierarchy of segmented images by partitioning an image into its constituent regions at hierarchical levels of allowable dissimilarity between its different regions. The hierarchy represents the continuous merging of similar, spatially adjacent, and/or disjoint regions as the allowable threshold value of dissimilarity between regions, for merging, is gradually increased.This chapter discusses in detail first the implementation of the HCS process, second the implementation details of how the HCS process is used for the presentation of multi-modal imaging data (MALDI and MRI) of a biological sample, third the implementation details of how the process is used as a perception aid for X-ray mammogram readers, and finally the implementation details of how it is used as an interpretation aid for the interpretation of Multi-parametric Magnetic Resonance Imaging (mpMRI) of the Prostate.

Mass spectrometry imaging tools in oncology.

MS imaging allows profiling and imaging of compounds directly from tumor tissue, tissue microarrays and tissue-engineered models of tumors. Methodologies for the quantitative analysis of localized/colocalized ion signals from a single cancer cell would be a major advance. Alternative methods of generating ions to matrix-assisted laser desorption ionization are increasingly employed. Desorption electrospray ionization has been used for the intraoperative diagnosis of human brain tumors and secondary ion MS imaging with cluster primary ion sources has been used for high spatial resolution imaging tumor sections. Extensive validation of the technique for the analysis of disease biomarkers is required, if imaging MS is to have a future role in the clinic.

MALDI-MS imaging for the study of tissue pharmacodynamics and toxicodynamics.

Pharmacodynamics and toxicodynamics are the study of the biochemical and physiological effects of therapeutic agents and toxicants and their mechanisms of action. MALDI-MS imaging offers great potential for the study of pharmaco/toxicodynamic responses in tissue owing is its ability to study multiple biomarkers simultaneously in a label-free manner. Here, existing examples of such studies examining anticancer drugs and topically applied treatments are described. Examination of the literature shows that the use of MS imaging in pharmaco/toxicodynamic studies is in fact quite low. The reasons for this are discussed and potential developments in the methodology that might lead to its further use are described.

MALDI-MSI and label-free LC-ESI-MS/MS shotgun proteomics to investigate protein induction in a murine fibrosarcoma model following treatment with a vascular disrupting agent.

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response biomarkers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate was examined in a mouse fibrosarcoma model (fs188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by combretastatin A4-phosphate and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-mass spectrometry imaging. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners.

Recombinant " IMS TAG" proteins--a new method for validating bottom-up matrix-assisted laser desorption/ionisation ion mobility separation mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) provides a methodology to map the distribution of peptides generated by in situ tryptic digestion of biological tissue. It is challenging to correlate these peptides to the proteins from which they arise because of the many potentially overlapping and hence interfering peptide signals generated. METHODS: A recombinant protein has been synthesised that when cleaved with trypsin yields a range of peptide standards for use as identification and quantification markers for multiple proteins in one MALDI-IMS-MSI experiment. Mass spectrometry images of the distribution of proteins in fresh frozen and formalin-fixed paraffin-embedded tissue samples following in situ tryptic digestion were generated by isolating signals on the basis of their m/z value and ion mobility drift time, which were correlated to matching peptides in the recombinant standard. RESULTS: Tryptic digestion of the IMS-TAG protein and MALDI-MS analysis yielded m/z values and ion mobility drift time for the signature peptides included in it. MALDI-IMS-MSI images for the distribution of the proteins HSP90 and vimentin, in FFPE EMT6 mouse tumours, and HSP90 and plectin in a fresh frozen mouse fibrosarcoma, were generated by extracting ion images at the corresponding m/z value and drift time from the tissue samples. CONCLUSIONS: The IMS-TAG approach provides a new means to confirm the identity of peptides generated by in situ digestion of biological tissue.

Detection of the epidermal growth factor receptor, amphiregulin and epiregulin in formalin-fixed paraffin-embedded human placenta tissue by matrix-assisted laser desorption/ionization mass spectrometry imaging.

The study of the expression and the tissue distribution of the tyrosine kinase drug-target epidermal growth factor receptor (EGFR) is of interest in oncology as a marker of potential efficacy of treatment. It has been reported, however, that the response rates to anti-EGFR drugs are poorly linked to its expression. Clinical studies have also revealed a patient response correlation with the expression levels of two EGFR ligands; amphiregulin and epiregulin. Here, we report the development of a matrix-assisted laser desorption/ionisation mass spectrometry imaging methodology for the study of EGFR, epiregulin and amphiregulin distribution in formalin fixed paraffin embedded human placental tissue and a comparison to expression patterns obtained by immunohiostochemistry. Using on-tissue digests and imaging of specific peptides, the tissue distribution of these proteins has been obtained down to 30 microm spatial resolution.

Introduction of a 20 kHz Nd:YVO4 laser into a hybrid quadrupole time-of-flight mass spectrometer for MALDI-MS imaging.

A commercial hybrid quadrupole time-of-flight mass spectrometer has been modified for high-speed matrix-assisted laser desorption ionisation (MALDI) imaging using a short-pulse optical technology Nd:YVO(4) laser. The laser operating in frequency-tripled mode (lambda = 355 nm) is capable of delivering 1.5-ns pulses of energy at up to 8 microJ at 5-10 kHz and 3 microJ at 20 kHz. Experiments to improve beam homogeneity and reduce laser speckle by mechanical vibration of the fibre-optic laser delivery system are reported along with data from trial and tissue imaging experiments using the modified instrument. The laser appeared to yield best results for MALDI-MS imaging experiments when operating at repetition rates 5-10 kHz. Combining this with raster imaging allowed images of rat brain sections to be recorded in 37 min. Similarly, images of the distribution of peptides in "on-tissue" digest experiments from tumour tissues were recorded in 1 h and 30 min rather than the 8-h acquisition time previously used. A brief investigation of targeted protein analysis/imaging by multiple reaction monitoring experiments "on-tissue" is reported. A total of 26 transitions were recorded over a 3-s cycle time and images of abundant proteins were successfully recorded.