In 2018, the research project ‘Identifying changes in proteome of single cells using imaging mass spectrometry’ received an award of over £135,000 under the lead of Professor Sir Doug Turnbull, who led the NIHR Newcastle BRC Neuromuscular Disease theme.
Understanding disease mechanisms and developing new treatments is a key priority for the Newcastle BRC and the assessment of patient tissue is crucial in this process. The project developed a new imaging mass cytometry (IMC) technique, applying it across a range of BRC themes and helping establishing it as an important ongoing tool for researchers. The project optimised the Hyperion system on muscle sections; a technique to detect up to 40 proteins in a sample. Prior to this, taking samples was a very sensitive procedure and involved looking down a microscope at proteins which are labelled with probes that fluoresce, meaning only the changes in four different proteins on a tissue sample could be detected.
Combining funding from the Medical Research Council (MRC), Wellcome Trust and Cancer Research UK, IMC played a central role in the evaluation of muscle biopsies from a life course study of age-related changes in muscle and enabled other studies to evaluate tissue from clinical trials. For example, comprehensive analysis of muscle biopsies from individuals through the life course and in patients with sarcopenia was one of the significant milestones of this research project.
The project ran for 36 months and has had a lasting impact. The team developed a catalogue of validated antibodies for use across the NIHR Newcastle BRC, including antibodies to cellular pathways for detection of individual cell types and for cell membranes. They were also able to optimise antigen retrieval and staining conditions for these antibodies in different tissue settings under different preservation conditions. Samples were used in the evaluation of clinical trials for long-term conditions and new clinical diagnostic pathways and new therapeutic approaches were developed.
With the help of the grant, Professor Andrew Filby and the team of Research Technical Professionals (RTPs) at the Newcastle University Flow Cytometry Core Facility (FCCF) set up the Hyperion system - an integral part of not only this project, but of countless others. To date, they have analysed tissues from nearly every part of the human body across several different developmental stages and diseases; many of which aligned to the BRC Themes. A key deliverable from this project was the creation of a database of validated antibodies for multiplexed tissue analysis. This wealth of information has been made available to BRC researchers and the wider community as an open access resource for those who contribute data and information to it. Currently, it stands at over 400 validated clones for key biomarkers and includes information on what types of tissues it works in, what experimental conditions are required and also includes example data. The database also includes information on clones that have not worked at all or in certain tissues as these data are of equal importance. Significantly, the information in the database also extends to other tissue imaging technologies that include the FCCFs latest multi-plexed imaging platform, the Miltenyi MACSima. The data obtained from these technologies supported by the antibody database has uncovered several cell types and spatial signatures that have relevance to human health and disease, feeding in potential novel biomarkers for lower lex tissue diagnostic tests.
The Hyperion Imaging System brings CyTOF technology together with imaging capability to allow the simultaneous detection of up to 40 different markers using IMC. Using the Hyperion allows analysis of tissue architecture and spatial relationships producing high dimensional image data from tissue sections.
The Hyperion Tissue Imaging unit allows for high dimensional analysis of tissue sections by mass cytometry. Tissue is analysed directly from the slide; preserving spatial relationships and eliminating the need to disassociate into single cell suspensions.
Imaging is achieved through laser ablation of sections stained with isotope-labelled antibodies and probes. Clouds of these rare earth metals are analysed by time of flight (TOF) cytometry and digitally reconstructed for in-depth image analysis.
The NU-FCCF is one of the Faculty of Medical Sciences (FMS) scientific facilities. It resides within the Newcastle University Biosciences Institute (NUBI) but supports research groups from right across the Faculty, University and a number of external collaborators and commercial companies. The initial seed funding from the NIHR Newcastle BRC to “pump prime” the Hyperion system both in terms of dedicated staff support, antibody validation and database construction has led to significant outcomes, impact and onward funding right across the BRC Themes, NUBI and beyond. This included using the system to analyse post-mortem lung tissue from COVID-19 patients as part of the UK Covid Immunology Consortium (UK-CIC). This project is a perfect example of how leading-edge technologies, RTPs from NUBI and clinical colleagues from the BRC Themes can work together to implement advanced analytical approaches to clinal questions that will, in time, generate significant patient benefit though new understandings of disease states that will underpin diagnosis, monitoring and therapy.
The Flow Cytometry Core Facility at Newcastle University