Laura M. Cole

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.

Instrumentation and software for mass spectrometry imaging—Making the most of what you've got

Whilst it might be desirable to be able to purchase an up to date mass spectrometry platform and dedicate it to mass spectrometry imaging, this is not the situation initially for many laboratories. There are a variety of methods by which existing mass spectrometers can be upgraded/adapted to perform mass spectrometry imaging using MALDI, DESI or LAESI as the means of generating ions. The focus of this article is on relatively low cost adaptations of existing instrumentation with suggestions made for performance enhancements where appropriate. A brief description of attempts to perform SIMS imaging on quadrupole time of flight mass spectrometers is also given. The required software is described with particular emphasis on freeware packages which can be used to display/enhance data. Requirements for data pre-processing prior or statistical analysis are discussed along with the use of MATLAB® for the analysis itself.

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.

Mass spectrometry imaging for the proteomic study of clinical tissue

Over the last decade, MALDI‐MS imaging has been used by researchers to explore areas of proteomics, lipidomics and metabolomics in samples of clinical origin for both targeted and global biomarker analysis. Numerous technological advancements in MS and clinical tissue MS imaging have been accomplished; hence, in this article we aim to critically discuss whether MS imaging has now in fact become a true champion of the ‘Omics Era’. In order to assess the potential for it to be routinely used in the clinical setting, it is pertinent to discuss some of its limitations, and to examine how these have been addressed by researchers. The key limitations of the technique we will discuss in this viewpoint article are as follows: sample throughput; relevance to patients, the availability of validated/standardised techniques; and integration with conventional pathology and other medical imaging techniques. Good progress has been made over the last 5 years in overcoming these limitations that had previously restricted the use of this technology in the clinical setting.

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.

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 μm spatial resolution.

MALDI-MSI for the analysis of a 3D tissue-engineered psoriatic skin model.

MALDI‐MS Imaging is a novel label‐free technique that can be used to visualize the changes in multiple mass responses following treatment. Following treatment with proinflammatory cytokine interleukin‐22 (IL‐22), the epidermal differentiation of Labskin, a living skin equivalent (LSE), successfully modeled psoriasis in vitro. Massons trichrome staining enabled visualization and quantification of epidermal differentiation between the untreated and IL‐22 treated psoriatic LSEs. Matrix‐assisted laser desorption ionization mass spectrometry imaging was used to observe the spatial location of the psoriatic therapy drug acetretin following 48 h treatments within both psoriatic and normal LSEs. After 24 h, the drug was primarily located in the epidermal regions of both the psoriatic and nonpsoriatic LSE models whereas after 48 h it was detectible in the dermis.

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.

Communication of medical images to diverse audiences using multimodal imaging

A study has been completed examining design issues concerning the interpretation of and dissemination of multimodal medical imaging data sets to diverse audiences. To create a model data set mouse fibrosarcoma tissue was visualised via magnetic resonance imaging (MRI), Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MSI) and histology. MRI images were acquired using the 0.25T Esaote GScan; MALDI images were acquired using a Q-Star Pulsar I mass spectrometer. Histological staining of the same tissue sections used for MALDI-MSI was then carried out. Areas assigned to hemosiderin deposits due to haemorrhaging could be visualised via MRI. In the MALDI-MSI data obtained the distribution sphingomyelin species could be used to identify regions of viable tumour. Mathematical ‘up sampling’ using hierarchical clustering-based segmentation provided a sophisticated image enhancement tool for both MRI and MALDI-MS and assisted in the correlation of images.