Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Autor: Cohen R; School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia; Colorectal Cancer Unit, St John of God Subiaco Hospital, Perth, Western, Australia. Electronic address: ryan.cohen@research.uwa.edu.au., Lee-Pullen T; Colorectal Cancer Unit, St John of God Subiaco Hospital, Perth, Western, Australia; School of Medicine, The University of Western Australia, Perth, Western, Australia. Electronic address: tracey.lee-pullen@uwa.edu.au., Miller TJ; Colorectal Cancer Unit, St John of God Subiaco Hospital, Perth, Western, Australia; School of Medicine, The University of Western Australia, Perth, Western, Australia. Electronic address: timothy.miller@uwa.edu.au., Meehan K; School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia. Electronic address: katie.meehan@uwa.edu.au., Fuller K; School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia. Electronic address: kathy.fuller@uwa.edu.au., McCoy MJ; Colorectal Cancer Unit, St John of God Subiaco Hospital, Perth, Western, Australia; School of Medicine, The University of Western Australia, Perth, Western, Australia. Electronic address: melanie.mccoy@uwa.edu.au.
Jazyk: angličtina
Zdroj: Methods (San Diego, Calif.) [Methods] 2023 Nov; Vol. 219, pp. 48-57. Date of Electronic Publication: 2023 Sep 21.
DOI: 10.1016/j.ymeth.2023.09.004
Abstrakt: Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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