P002 Methodological development of single-cell phenotyping and spatial analysis of intestinal leukocyte, stromal and epithelial cell populations in inflammatory bowel disease formalin fixed paraffin embedded tissue by Hyperion imaging mass cytometry
Lamb, C.A.(1,2);Doyle, J.(1);Hulme, G.(3);Cooke, K.(1);Au-Yeung, A.(4);Ojeda-Garcia, J.(3);Fuller, A.(3);McDonald, D.(3);Mansfield, J.C.(1,2);Kirby, J.A.(1);McBride, J.(4);Speight, R.A.(1,2);O’Gorman, W.(4);Filby, A.(3);
(1)Newcastle University, Translational and Clinical Research Institute, Newcastle upon Tyne, United Kingdom;(2)Newcastle upon Tyne Hospitals NHS Foundation Trust, Department of Gastroenterology, Newcastle upon Tyne, United Kingdom;(3)Newcastle University, Flow Cytometry Core Facility and Innovation- Methodology and Application Research Theme- Biosciences Institute- Faculty of Medical Sciences, Newcastle upon Tyne, United Kingdom;(4)Genentech Inc, Department of OMNI Biomarker Development, South San Francisco, United States
Background
Conventional cellular phenotyping of intestinal cell populations by multi-parameter fluorescence cytometry is reliant on collection of fresh tissue for immediate enzymatic or mechanical disaggregation, or cryopreservation of samples. These factors limit widescale use of tissue for research, increase cost, and time for sample collection or preparation. Due to tissue disaggregation suspension cytometry does not provide data regarding spatial localisation of cells in tissue. Formalin fixed paraffin embedded (FFPE) tissue is widely collected at endoscopy and surgery for clinical histopathological assessment, and can be stored and transported at room temperature.
Methods
We aimed to develop a method for analysing FFPE intestinal tissue using Imaging Mass Cytometry (IMC) in combination with an analysis pipeline for cellular phenotyping and spatial characterisation that preserved multi-parameter, high dimensional phenotyping capabilities normally only afforded by suspension methodologies. FFPE blocks were accessed following written informed consent in accordance with research and ethics committee approval. Carrier-free antibodies specific to cell subsets of interest were selected based on conventional suspension fluorescence cytometry and immunohistochemistry data.
Results
Antibodies were conjugated to metal isotopes. Antigen retrieval and antibody dilution was optimised on 4µm tissue sections using Tris-EDTA pH 9 initially by immunofluorescence then in multiple assays by Hyperion (Fluidigm) IMC (Figure 1A). An analysis pipeline was developed based on the “Bodenmiller approach” using a combination of R, Python and MATLAB packages: CellProfiler and ilastik to segment single cells, and ImaCyte to explore the resident phenotypes and cellular neighbourhoods in diseased and healthy tissues. A staining panel with 25 antibodies was optimised to identify stromal, epithelial and leukocyte populations. Training algorithms allowed computational segmentation of nuclear, cytoplasmic and non-cellular regions (Figure 1B), cell mask, segmentation and spatial analysis (Figure 1C), and t-SNE (Figure 1D). Representative three parameter images created in MCD viewer (Fluidigm) are shown in Figure 1E to demonstrate cell populations and spatial localisation.
Conclusion
Quantifiable, multiparameter cellular phenotyping with spatial visualisation can be undertaken with FFPE intestinal tissue using IMC. Due to the existence of archival healthcare samples, the ease of tissue acquisition, processing and storage of FFPE specimens this provides a valuable resource for investigation, including mechanisms of disease pathogenesis, molecular biomarker discovery, and longitudinal pharmacodynamic analysis in clinical trials.