Introduction
‘A picture is worth a thousand words’ is an idiom from the English language (‘borrowed’
from on old Chinese proverb) that conveys the notion that a complex idea can be succinctly
and fully described by a single image. Never has this expression been truer than in
the clinical and pharmaceutical arenas. Enormous strides have been made by the scientific
community in the evolving field of biomedical imaging with the aim of representing
and/or quantifying aspects of disease and drug action by using tools such as radiography
[1], MRI [2, 3] PET [4], and ultrasound [5]. Yet linking the phenotypical data generated
by these systems to the genome is a challenging task. Identifying the link between
the mechanism of disease or failed drug response to the genome of an individual is
difficult, because central pieces of information are missing. However, imaging mass
spectrometry (IMS) can overcome this issue. IMS aims to detect the molecular constituents
of the tissue; these can then be correlated with genome-related characteristics, such
as gene expression patterns and possible mutations, and ultimately provide a phenotypic
molecular link to the complex disease biology. The big data technology of IMS can
generate spatial information of thousands of metabolites and proteins from within
a tissue, facilitating a deeper understanding of the connections between the genome,
phenotypic characteristics and the biological response. It is a technology that has
the potential to serve as a segue between gene expression and observed biological
signal.
Image analysis has been a focus of mass spectrometry for more than 40 years since
early studies using secondary ion mass spectrometry (SIMS) [6]. Among the several
ionization techniques, matrix-assisted laser desorption ionization (MALDI) imaging
mass spectrometry is the leader for analyzing molecular distributions within tissues
[7]. MALDI IMS is capable of mapping biomolecules of interest at high spatial resolution
(~ 1 μm), and high sensitivity. It can be employed to image a broad variety of molecular
classes, from low-molecular-weight metabolites, lipids (> 1 kDa) and proteins [8,
9]. The unique ability of this technique to reveal these ionized molecular entities,
while retaining the spatial information for multiple molecules in one measurement,
makes histology-directed MALDI IMS a powerful tool for clinical applications and genome-based
personalized medicine [10]. Furthermore, desorption electrospray ionization (DESI)
[11] is an ionization technique that has the capability of direct solid surface sampling
under open ambient conditions. DESI has the advantages of ambient ionization methods
and combined with MALDI, hundreds-to-thousands of molecules can be evaluated simultaneously
and their spatial distribution can be visualized from within the same tissue section
(Table 1). Consequently, the molecular changes in a tissue can be accurately studied,
correlated to images and cellular features generated by traditional histology, and
the pathogenic mechanisms of a certain disease can be visualized and identified, leading
to the potential discovery of new biomarkers [8].
Table 1
Imaging mass spectrometry ionization techniques: application, advantages, and disadvantages
[8, 9, 11].
Application
Ionization
Advantages
Disadvantages
Tissue molecular imaging
MALDI
• Label-free analysis• High-sensitivity• Use over a broad mass range• High-spatial
resolution (≤ 1 μm)• Application on both formalin-fixed paraffin-embedded (FFPE) tissue
microarrays and on fresh tissue samples.
• Low throughput• Sample preparation can lead to spatial dislocation or chemical modifications•
Matrix dependent analysis
DESI
• Label-free analysis• Ambient ionization method• Direct solid surface sampling• Multiple
charged ions• Minimal sample preparation• Soft ionization method• Generally less costly
upon comparison to MALDI
• Imaging in the low-mass region—limited use for proteins• Poorer spatial resolution
compared to MALDI• Solvent dependent
The pharmaceutical industry has taken advantage of the development of IMS to enable
an array of high-throughput screening modalities for pharmaceutical assessments [12].
IMS can provide reliable, label-free qualitative and quantitative distribution information
for a drug of interest and its subsequent biotransformed metabolites [13]. This information
can be used to determine and understand the pharmacokinetic (PK) properties of a drug,
its penetration into tissue, and to assess drug efficacy and potential toxicity [14].
This makes IMS a powerful, yet cost-effective technology because distribution studies
can be performed earlier in the drug discovery process without any requirement for
radiolabeled standards. Of critical importance, IMS separately maps and differentiates
drug from its metabolites, rather than tracking just a radiolabeled parent drug [14–16].
At this 1-day symposium, the Department of Environmental Health Sciences (DEHS) at
the Yale School of Public Health brought together leaders in IMS to discuss recent
developments, limitations, and future needs, and to increase awareness of this growing
and important field. During his opening remarks, Dr. Vasiliou highlighted the potential
of IMS to define the molecular basis of diseases, to provide insights into mechanisms,
and to integrate tissue morphology at the molecular level. Thereafter, talks by an
impressive assembly of thought-leaders in IMS illustrated the potential of unbiased
tissue imaging to deliver a new level of understanding of pathophysiological processes
at the molecular level. The symposium concluded with a round-table discussion, chaired
by Dr. Mark Duncan, on some of the more practical issues in IMS such as current bottlenecks
and future opportunities. The presentations and discussions at the symposium underscored
the great potential of IMS. With the intention of bringing IMS to the larger scientific
community, the DEHS has committed significant resources to the acquisition of equipment
and expertise that will allow the further development and application of IMS techniques.
MALDI imaging mass spectrometry (IMS): recent technological advances
The first talk of the symposium was given by a pioneer of the field, Dr. Richard Caprioli,
Professor of Biochemistry and Director of the Mass Spectrometry Research Center at
Vanderbilt University. His opening remarks gave a historical overview of MALDI IMS
technology and emphasized its advantages. Dr. Caprioli explained how MALDI IMS employs
desorption of molecules by direct laser irradiation to map the location of specific
molecules from fresh frozen or formalin-fixed tissue sections without the need to
target specific reagents, such as antibodies [17]. Dr. Caprioli championed the major
benefits of the histology-directed approach (that has been developed by his group)
over conventional staining and microscopic methods. This technology is an addition
to the histologist’s toolbox, not a replacement. By integrating microscopy with MALDI
IMS, this application is almost limitless and could be used in a variety of biologically
and medically relevant research projects. Dr. Caprioli highlighted studies in diabetic
nephropathy involving both proteins and lipids and the differentiation of benign skin
lesions from melanomas [17, 18]. In addition, Dr. Caprioli’s group has applied IMS
to drug targeting and metabolic studies in specific organs and in intact whole animal
sections following drug administration [19]. Recent technological advances were also
described for sample preparation to improve metabolite extraction and instrument performance
to achieve images at high spatial resolution (1–10 μm) and at high speeds so that
a typical sample tissue, once prepared, can be imaged in minutes [20]. Instrumentation
used in these studies included both MALDI fourier transform ion cyclotron resonance
(FTICR) and MALDI time-of-flight (TOF) mass spectrometers. Applications utilize tandem
mass spectrometry (MS/MS), ultra-high mass resolution, and ion accumulation devices
for IMS studies. Finally, new biocomputational approaches were discussed that are
required to handle the high-data dimensionality of IMS, and also ‘image fusion’ for
predictive integration of mass spectrometry (MS) images with microscopy and other
imaging modalities [21].
Dr. Kevin Schey, Professor of Biochemistry, Ophthalmology and Visual Sciences, at
Vanderbilt University, discussed the application of IMS to study a range of molecular
classes, such as proteins, lipids, and metabolites in ocular tissues. Ocular tissues
provide an ideal medium to demonstrate the utility of the technique where morphological
features are on the scale of single cells. For example, molecular profiles can be
produced in retina pigment epithelium. Moreover, a range of diseases affect the various
ocular tissues, including glaucoma, age-related macular degeneration, cataract, and
corneal cataract. Dr. Schey illustrated how IMS is being actively applied to derive
mechanistic information that enhances understanding about the molecular underpinnings
of disease in these tissues as well as aging mechanisms. IMS data from optic nerve,
retina, lens, and cornea were presented with special attention to diseases affecting
these tissues [22]. Data from both animal models of disease and human tissues were
discussed, as well as key methodological details for successful imaging ocular tissues
[23–25]. Dr. Schey’s work, in collaboration with Dr. Vasiliou, on a corneal haze phenotype
in Aldh3a1-null mice presented the first genetic animal model of cellular-induced
corneal haze due to the loss of a corneal crystallin [24]. This work clearly showed
how IMS can provide deeper understanding for the genome, linking the disease phenotype
with genetic changes.
The broad range of IMS applications was reinforced even more by Dr. Andrén, Professor
at Uppsala University, who showed novel ways to interrogate the actions of neurotransmitters,
their precursors and metabolites, in the brain chemical network and neuronal signal
transmission [26]. Changes in neurotransmitter concentrations are associated with
numerous normal neuronal processes, such as sleep and aging, and in several disease
states, including neurological disorders (e.g., Parkinson’s and Alzheimer’s disease),
depression, and drug addiction. Dr. Andrén uses knowledge about the relative abundance
and spatial distribution of neurotransmitters in the brain to provide insights into
these complex neurological processes and disorders. At present, researchers rely on
indirect histochemical, immunohistochemical, and ligand-based assays to detect small-molecule
transmitter substances or on tissue homogenates analyzed by high-performance liquid
chromatography analysis. Current neuroimaging techniques have very limited capacities
to directly identify and quantify neurotransmitters from brain sections. MALDI IMS
can perform analyses directly on the surface of a tissue section, establishing itself
as a powerful in situ visualization tool for measuring abundance and spatial distribution
of endogenous and pharmaceutical compounds, lipids, peptides, and small proteins.
A novel reactive MALDI matrix, recently developed by Dr. Andrén’s group, selectively
targets the primary amine group on neurotransmitters, metabolites, and neuroactive
substances while also functioning as a matrix to enable ionization [27]. However,
the limitation of using such a reactive matrix to study the full molecular pathways
of, for example, dopaminergic or serotonergic biosynthesis and metabolism is its limitation
to target all downstream dopamine metabolites derived from monoamine oxidase (MAO)
or catechol-O-methyltransferase (COMT) enzymes. The majority of small molecule neurotransmitters,
such as catecholamines, amino acids, and trace amines, possess phenolic hydroxyl and/or
primary or secondary amines which are strong nucleophilic groups. Dr. Andrén’s laboratory
has therefore developed a new reactive matrix that can selectively target and charge-tag
both phenolic and primary amine groups, thus enabling MALDI IMS of both MAO and COMT
downstream metabolites, focusing on a nucleophilic aromatic substitution reaction
with such functional groups. Using this new reactive matrix, they were able to detect
and map the localization of most of the neurotransmitters and metabolites involved
in the dopaminergic and serotonergic network in a single brain tissue section. This
work showed a novel methodology that assists with metabolite identification through
the selectivity of the reaction. The sensitivity and specificity of this imaging approach
to neurochemicals has great potential for many diverse applications in neuroscience,
pharmacology, drug discovery, neurochemistry, and medicine.
Visualizing drug disposition in tissue
A major focus of the symposium was the application of MALDI IMS to map the distribution
of a variety of therapeutic molecules across a tissue section of interest and to assess
their biological impact.
Current president of the Imaging Mass Spectrometry Society and director of US Imaging
MS, at GlaxoSmithKline (GSK), Dr. Castellino, discussed how MALDI IMS technology has
taken their research beyond “plasma-centric” studies and allowed for direct mapping
of molecular changes in tissue associated with drug pharmacology, disposition, and
disease pathogenesis. Delivering safe and efficacious drugs is tied to the ability
to understand complex mechanistic relationships between molecular initiation events
of pharmacologically active compounds and the cascade of subsequent biological consequences.
Because the delivery of drugs to their intended target, and avoidance of unintended
targets, is a critical first step, IMS can directly guide improvements and innovation
in delivery strategies by mapping the target tissue selectivity [28]. Furthermore,
tissue correlations can be directly made to plasma PK or lead to improved pharmacodynamic
understanding. Dr. Castellino’s group has explored the use of MALDI IMS to investigate
the distribution of drugs and their metabolites, as well as endogenous compounds,
in a wide variety of target tissues in support of numerous therapeutic areas and in
all drug discovery and development stages [29, 30]. The IMS methodology allows for
the co-registration of drug analytes in tissue distributions with histology images,
thereby integrating chemical structures with tissue morphology. Furthermore, this
imaging modality offers the potential to further our mechanistic understanding of
drug disposition, disease progression, and pharmacology (including toxicology) by
providing snap shots of temporal and causal changes [31]. Dr. Castellino continued,
that while MALDI IMS is primarily being employed to determine the tissue distribution
of drugs and their metabolites, it has become evident that more detailed understanding
of biological systems can be gained by including the changes in endogenous compound
distribution as a function of disease and pharmacology. Closing his presentation,
Dr. Castellino discussed the importance of suitable software tools and improved data
handling methods needing to be developed alongside analytical progress in order for
the full potential of MALDI IMS to be realized.
Dr. Richard Goodwin, a principal scientist for Drug Safety and Metabolism at AstraZeneca
(AZ) and head of mass spectrometry imaging, presented the challenges faced for drug
discovery and development; it is a lengthy, high risk, and competitive business that
can take a decade to progress; moreover, billions of dollars are required to move
a new medicine to market. Dr. Goodwin discussed how IMS could help mitigate some of
the primary reasons for drug attrition, specifically around lack of efficacy and toxicological
or clinical safety risk. IMS is now demonstrating impact on drug discovery programs
and helping reduce later stage compound attrition. It provides insights into the biodistribution
of compounds, while simultaneously generating data on pharmacodynamic biomarkers.
Dr. Goodwin presented data from AZ that showed how the use of a range of multimodal
imaging techniques improves understanding about compound efficacy, safety, and targeted
drug delivery [32, 33]. Investigating histopathological-targeted drug-induced toxicity
is now readily achieved using high-spatial resolution and high-mass resolution IMS.
Dr. Goodwin outlined how a Cancer Research UK Grand Challenge consortium are seeking
to use multimodal IMS to offer new insights into tumor metabolism and to help develop
new, more effective medicines and therapy combinations. The $20 million project led
by Professor Bunch at the National Physical Laboratory UK (in collaboration with world
leading oncology biologists, IMS technologists, and AZ) will utilize data similar
to that shared at the symposium. IMS can help identify metabolite changes consistent
with the biomarker changes in the tumor and show changes in metabolites as PD biomarkers,
hence providing valuable new insights into the pathway and drug combinations. The
next hurdle is how to effectively mine multimodal imaging data. Recent strategies
on data processing and visualization as well as data mining algorithms were outlined
[34]. In his closing remarks, Dr. Goodwin highlighted the challenges and opportunities
arising from the significant quantities of molecular imaging data generated, from
a cellular to patient level.
Dr. Sheerin Shahidi-Latham, Head of Metabolomics and Imaging MS, Department of Drug
Metabolism and Pharmacokinetics at Genentech Inc., also discussed the advantages of
the applied use of MALDI IMS. In the pharmaceutical industry, obtaining information
about the absorption, distribution, metabolism, and elimination (ADME) of a new chemical
entity via a PK study in a preclinical animal model is often the first step towards
understanding the in vivo properties of a drug-like molecule in humans. Traditionally,
much of this ADME work has been supported by liquid-chromatography coupled to mass
spectrometry. In the case of tissue distribution, the organs are excised and homogenized
in order to accommodate this analytical workflow, thereby effectively eliminating
any spatial information. Dr. Shahidi-Latham emphasized that MALDI IMS has gained prominence
since it provides a robust, label-free detection of drug and metabolites while preserving
spatial localization within tissue sections of interest. Additionally, the use of
high-resolution mass spectrometers has provided the opportunity for simultaneous detection
of subsequent pharmacodynamic (PD) responses within a single image acquisition. Dr.
Shahidi-Latham discussed how the ability to assess PK/PD relationships in a label-free,
in situ context has proven invaluable to the early lead optimization efforts that
take place in the drug discovery setting. Similarly, uncovering the perpetrator of
adverse effects often associated with histopathological assessments in the preclinical
development phase has also improved their understanding about the mechanisms of toxicity
and can provide useful information for the redesign of a back-up molecule. The presentation
provided a synopsis of the advantages of IMS, as well as the technical challenges
and opportunities in the context of the pharmaceutical industry. Dr. Shahidi-Latham
presented data from her work at Genentech, which included MALDI IMS of dosed tissues
in support of drug efficacy, PK/PD, and effective delivery evaluations, and highlighted
the simultaneous detection of drug, metabolites, and endogenous components attainable
from a single imaging run [35–39]. Moreover, examples demonstrating the utility of
imaging MALDI IMS for toxicity screening were presented. Its complementary use with
autoradiography analyses were discussed, including ocular drug distribution and whole-body
drug disposition studies [15].
Bioinformatics platform for large-scale mass spectrometry imaging data
The constant drive towards integrating complex and large-scale datasets has generated
the need to develop new tools to process this information [40]. Dr. Kirill Veselkov,
Lecturer at Imperial College London, discussed how managing, analyzing, and interpreting
these data are a challenge and a major barrier to their clinical translation. IMS
augments digital pathologic analysis with highly robust big data on cellular metabolic
and proteomic molecular content, generating a staggering amount of unrefined data
(tens to hundreds of gigabytes of data per tissue section). Existing data analysis
solutions for IMS rely on a set of heterogeneous bioinformatics packages that are
not scalable for the reproducible processing of large-scale (hundreds to thousands)
biological sample sets. In this talk, Dr. Veselkov presented a computational platform
(pyBASIS) capable of optimized and scalable processing of IMS data for improved information
recovery and comparative analysis across tissue specimens using machine learning and
related pattern recognition approaches. The proposed solution also provides a means
of seamlessly integrating experimental laboratory data with downstream bioinformatics
interpretation and analyses, resulting in a truly high-throughput system for translational
IMS.
The symposium concluded with a round-table discussion, chaired by Dr. Mark Duncan,
where the attendees discussed the practical challenges and future directions of IMS.
The attendees not only agreed on the potential of IMS to provide previously inaccessible
insights into molecular events at the tissue level, but also highlighted the cost
and complexity of both the science and the technology that underpins these studies.
Rather than a routine core service, it was agreed that designing meaningful studies,
performing exacting sample handling, generating and interpreting complex data, and
maintaining high-end instrumentation requires a substantial, highly collaborative
interaction between all stakeholders.