Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue
Hart, Claire A
Carey, C. D.
Vincent, A. E.
Greaves, L. C.
Brown, Michael D
Clarke, Noel W
Turnbull, D. M.
AffiliationGenito Urinary Cancer Research Group, Division of Cancer Sciences, Oglesby Cancer Research Building, University of Manchester, Manchester, M20 4GJ, UK.
MetadataShow full item record
AbstractAdvances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.
CitationSachdeva A, Hart CA, Carey CD, Vincent AE, Greaves LC, Heer R, et al. Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue. Vol. 12, Scientific Reports. Springer Science and Business Media LLC; 2022.
- Multiplexed Imaging for Immune Profiling on Human FFPE Material.
- Authors: Mezheyeuski A, Strell C
- Issue date: 2021
- Manual Immunofluorescence of Formalin-Fixed Paraffin-Embedded Human Tumor Tissues.
- Authors: Nersesian S, Boudreau JE
- Issue date: 2022
- Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens.
- Authors: Surace M, DaCosta K, Huntley A, Zhao W, Bagnall C, Brown C, Wang C, Roman K, Cann J, Lewis A, Steele K, Rebelatto M, Parra ER, Hoyt CC, Rodriguez-Canales J
- Issue date: 2019 Jan 21
- Multiplex Immunofluorescence Assays.
- Authors: Francisco-Cruz A, Parra ER, Tetzlaff MT, Wistuba II
- Issue date: 2020
- Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer.
- Authors: Mantsiou A, Makridakis M, Fasoulakis K, Katafigiotis I, Constantinides CA, Zoidakis J, Roubelakis MG, Vlahou A, Lygirou V
- Issue date: 2020 Jul 2