Abstract 203: Automated, high throughput mass spectrometry imaging of serial sections for investigation of tumor heterogeneity in three dimensions
Authors: Jones Emrys A. , Henderson Fiona , Coope David , Denbigh Joanna , Pringle Steven , McMahon Adam , Williams Kaye 
Published: July 2017

Abstract
For the purpose of investigating the potential of automation and high throughput mass spectrometry imaging, we have combined a desorption electrospray ionisation (DESI) MS system with a robotic slide loader. The analysis of tissue sections on multiple slides could allow, for example, large patient cohorts to be analysed with almost no input from the operator. Here we present another use of such a system, the mapping and visualisation of tissues in 3D from serial sections.
All experiments were carried out on a Waters Xevo G2-XS Q-ToF equipped with a Prosolia (Indianapolis) 2D DESI stage. The automated slide loader and modified plate holder were provided by Prior Scientific (Cambridge). As an ambient ionisation technique, the DESI sampling procedure occurs outside of the vacuum of the instrument and is therefore suited to accommodate slide handling devices. A modified top plate was created that, once in place, allows the Prior PL200 microscope slide loader to load slides from the holding cassettes to the stage and back. A snapshot of the slide being taken and object definition algorithms is used to identify the location of the tissue(s) on the slide. These co-ordinates are then written to the experiment file and the acquisition commences. This process will continue until all slides are analysed.
Recent technological advances have led to the time taken for a single MS tissue image being reduced from the region of hours to minutes. This then opens up the possibility of automatic sample loading and high throughput usage. Fifteen sections taken at 120µm steps through a human glioma were analysed automatically by the DESI system. Data was collected in both positive and negative ion mode from the same tissue to increase the molecular coverage. Sections taken from between those use for the imaging were used for H&E staining and CA-9, Ki67 and SCD immunohistochemistry staining. Once all sections were analysed, recently developed software allowed for the reconstruction of the sections into a three dimensional representation of the chemically distinct regions within the whole tissue volume. This process which involves peak picking, object alignment and rotation as well as a non negative matrix factorisation step is all done automatically. When these regions identified from the 3D mass spectrometry mapping are compared to the CA-9 and Ki67 IHC stains the regions of hypoxia and proliferation clearly correlate indicating that mass spectrometry imaging could be a useful tool for the determination of hypoxia within excised tissue. Further to these identified regions was the discovery of molecular signals that were only present on the boundary between the hypoxic and normoxic regions; the identities and roles of which are currently under investigation.
Faster and automated mass spectrometry imaging allows whole tissues to be chemically mapped in three dimensions in a few hours providing insights into tumour heterogeneity.
Citation Format: Emrys A. Jones, Fiona Henderson, David Coope, Joanna Denbigh, Steven Pringle, Adam McMahon, Kaye Williams. Automated, high throughput mass spectrometry imaging of serial sections for investigation of tumor heterogeneity in three dimensions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2017-203