Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue

Ashwin Sachdeva*, Claire A. Hart, Christopher D. Carey, Amy E. Vincent, Laura C. Greaves, Rakesh Heer, Pedro Oliveira, Michael D. Brown, Noel W. Clarke, Doug M. Turnbull

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Advances 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.

Original languageEnglish
Article number6660
JournalScientific Reports
Volume12
Issue number1
Early online date22 Apr 2022
DOIs
Publication statusPublished - 22 Apr 2022

Keywords

  • Electron Transport Complex IV
  • Fluorescent Antibody Technique/methods
  • Formaldehyde
  • Humans
  • Male
  • Oxidative Phosphorylation
  • Paraffin Embedding
  • Prostate/diagnostic imaging
  • Prostatic Neoplasms/diagnostic imaging
  • Tissue Fixation

Research Beacons, Institutes and Platforms

  • Manchester Cancer Research Centre

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