Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment
Ryan Cohen1, Tracey Lee-Pullen2, Timothy J Miller2
1School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia; Colorectal Cancer Unit, St John of God Subiaco Hospital, Perth, Western, Australia.
Methods (San Diego, Calif.)
|September 23, 2023
Summary
This study optimized a five-color multiplex immunofluorescence panel for colorectal cancer research. The method accurately characterizes T cell populations and their spatial distribution in the tumor microenvironment.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- The tumor microenvironment (TME) is crucial for understanding host-tumor interactions and anti-tumor immune responses.
- Immune biomarkers in the TME are superior to traditional staging for predicting colorectal cancer recurrence.
- Multiplex immunofluorescence (mIF) allows detailed immune cell classification and spatial characterization within tumor tissues.
Purpose of the Study:
- To optimize an automated, five-color multiplex immunofluorescence panel for characterizing T cell populations in colorectal cancer.
- To validate the use of tyramide signal amplification (TSA)-based mIF for translational research.
- To establish a reproducible workflow for mIF in colorectal adenocarcinoma tissue analysis.
Main Methods:
- Development of a five-color TSA-based mIF panel using antibodies against CD3, CD8, CD103, and cytokeratin.
- Optimization of automated slide staining, including primary antibody titration and monoplex assay development.
- Detailed protocols for antibody stripping, horseradish peroxidase inhibition, staining sequence determination, and fluorophore assignment.
- Image acquisition using a standard fluorescence microscope slide scanner and management of spectral crosstalk.
- Digital image analysis for cell characterization and spatial distribution assessment within the TME.
Main Results:
- Successful optimization of an automated, five-color mIF panel for colorectal cancer.
- Demonstration of reproducible staining and characterization of T cell populations (CD3+, CD8+, CD103+).
- Establishment of a workflow enabling spatial analysis of immune cells within the tumor microenvironment.
Conclusions:
- Automated mIF with TSA provides a consistent, cost-effective, and accurate method for analyzing immune cells in colorectal cancer.
- This optimized panel and workflow facilitate detailed spatial characterization of the TME, crucial for understanding anti-tumor immunity.
- The described methodology supports translational research by enabling researchers to utilize mIF with standard equipment.
