Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An
Adrien Guillot1, Marlene S Kohlhepp1, Alix Bruneau1
1Department of Hepatology & Gastroenterology, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany.
Insights
A new, cost-effective multiplex immunostaining method enables detailed analysis of immune cells in solid organs. This technique reveals insights into tissue organization and macrophage heterogeneity in liver diseases, including hepatocellular carcinoma (HCC).
Area of Science:
- Immunohistochemistry and Molecular Pathology
- Cancer Biology and Immunology
- Organ-Specific Pathophysiology
Background:
- Tumor microenvironment complexity is increasingly understood at the single-cell level.
- Immune cell composition, spatial distribution, and histology offer diagnostic and therapeutic insights.
- Multiplex immunostaining on formalin-fixed paraffin-embedded (FFPE) tissues is valuable but technically challenging.
Purpose of the Study:
- To develop a simple, cost-effective, and reproducible multiplex immunostaining protocol for FFPE tissues.
- To enable flexible multiplexing (>12 antibodies) applicable to routine histology laboratories.
- To characterize the hepatic immune microenvironment in preclinical models and human liver diseases.
Main Methods:
- Developed a novel, simplified multiplex immunostaining protocol optimized for solid organs (liver, intestine, lung, kidney) in mice and humans.
- Protocol requires minimal specialized equipment and reagents for broad accessibility.
- Combined multiplex immunostaining with automated whole-slide image analysis for comprehensive characterization.
Main Results:
- Successfully applied the method to analyze hepatic immune microenvironments in mouse models of liver fibrosis, steatohepatitis, and hepatocellular carcinoma (HCC).
- Characterized immune cell interactions within human patient samples of chronic liver diseases.
- Revealed significant macrophage heterogeneity in the liver across premalignant conditions and HCC.
Conclusions:
- The developed multiplex immunostaining protocol is highly flexible, cost-effective, and reproducible for FFPE tissues.
- This method provides valuable insights into tissue organization and immune-parenchymal cell interactions.
- The findings underscore the importance of macrophage heterogeneity in liver disease progression and HCC.
Abstract:
Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune-parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.
