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Updated: Jul 30, 2025
Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
Published on: January 21, 2019
Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic
Fernanda Marconi Roversi1,2, Maura Lima Pereira Bueno3, Juliete Aparecida Francisco da Silva4
1Hematology and Transfusion Medicine Center, University of Campinas/Hemocentro-Unicamp, Rua Carlos Chagas, 480 - Cidade Universitária Zeferino Vaz - Barão Geraldo Campinas, São Paulo, CEP: 13083-878, Brazil. feroversi@gmail.com.
Insights
This study presents an optimized multiplex-fluorescence staining technique for formalin-fixed paraffin-embedded tissues. The method enhances biomarker discovery for improved tumor diagnosis and prognosis.
Area of Science:
- Oncology
- Biotechnology
- Medical Imaging
Background:
- Neoplastic cell studies yield tumor biomarkers crucial for early diagnosis, therapy, and prognosis.
- Immunofluorescence (IF) is a high-throughput imaging technique for cell characterization, preserving tissue architecture.
- IF on formalin-fixed paraffin-embedded (FFPE) tissues faces challenges like autofluorescence and non-specific binding.
Purpose of the Study:
- To develop a multiplex-fluorescence staining technique for high-contrast, high-quality multicolor imaging of biomarkers.
- To optimize IF procedures for FFPE tissues, reducing autofluorescence and enabling simultaneous antibody use.
- To achieve super-resolution imaging for precise antigen localization.
Main Methods:
- Developed an optimized multiple-immunofluorescence staining protocol.
- Applied the technique to FFPE neoplastic biopsies (appendix, lymph node, bone marrow) and 3D-coculture systems.
- Focused on reducing sample autofluorescence and enabling simultaneous antibody detection.
Main Results:
- Successfully reduced sample autofluorescence.
- Enabled simultaneous antibody staining on the same FFPE sample.
- Achieved super-resolution imaging for precise antigen localization in various neoplastic tissues and 3D models.
Conclusions:
- The optimized multiplex-IF method is a powerful tool for understanding tumor cell complexity and spatial localization.
- It aids in identifying predictive and prognostic biomarkers and immunologic phenotypes from limited samples.
- This protocol enables tumor microenvironment profiling, aiding research on cellular crosstalk, niches, and predictive biomarker discovery for neoplasms.
Abstract:
The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.
