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Updated: Dec 27, 2025
Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment
Published on: March 25, 2020
Multiplexed immunohistochemistry for immune cell phenotyping, quantification and spatial distribution in situ
Angela Vasaturo1, Jérôme Galon2
1Ultivue, Inc, Cambridge, MA, United States.
Insights
Multiplex immunofluorescence (IF) assays provide deep profiling of the tumor immune microenvironment. This method aids in identifying immune biomarkers and stratifying patients for immunotherapy by analyzing immune cell infiltration and PD-L1 expression.
Area of Science:
- Immunology
- Oncology
- Biomarker Discovery
- Digital Pathology
Background:
- Characterizing the immune microenvironment is crucial for identifying prognostic and predictive immune biomarkers.
- Advances in tissue imaging, particularly multiplex immunohistochemistry (IHC), enable quantitative assessment of immune cell phenotypes and spatial distribution.
- Multiplex immunofluorescence (IF) offers a powerful approach to analyze immune infiltration and protein expression within the tumor microenvironment.
Purpose of the Study:
- To detail a protocol for multiplex immunofluorescence (IF) assay on FFPE tumor tissue for immune profiling.
- To characterize immune infiltration and PD-L1 expression using a five-marker panel (CD8, CD68, PD-L1, CK, SOX10).
- To provide an overview of current multiplex image analysis solutions for digital pathology.
Main Methods:
- Application of multiplex immunofluorescence (IF) technology to FFPE tumor tissue.
- Utilized a five-protein marker panel: CD8, CD68, PD-L1, CK, and SOX10.
- Employed optimized assay workflow compatible with existing instrumentation and routine digital pathology software for image analysis.
Main Results:
- The multiplex IF assay enables quantitative assessment of immune cell phenotypes and functional orientation within the tissue context.
- Facilitates analysis of marker expression dynamics, co-localization, and co-expression across the entire tissue.
- The UltiMapper™ I/O PD-L1 multiplex assay provides a fast and optimized workflow from sample preparation to image analysis.
Conclusions:
- Deep profiling of the tumor immune microenvironment using multiplex IF can guide the development of immune checkpoint strategies.
- This approach aids in better patient stratification for immunotherapy by identifying those likely to benefit.
- Multiplex IF technology enhances the understanding of immune infiltration and PD-L1 expression for precision oncology.
Abstract:
It is increasingly recognized that a deep characterization of the immune microenvironment is required for the identification of prognostic and predictive immune biomarkers. Recent advances in the field of tissue imaging resulted in the development of fluorescence multiplex IHC technologies enabling quantitative assessment of immune phenotypes and functional orientation of immune cells in a way similar to flow cytometry, while simultaneously providing tissue context and spatial distribution. Multiplex immunofluorescent technology to FFPE tumor tissue is applied to characterize immune infiltration and PD-L1 expression. A panel consists of five protein markers: CD8, CD68, PD-L1, CK, and SOX10. The assay workflow is fast, optimized and compatible with existing instrumentation. The resulting images can be analyzed with routinely used software for digital pathology enabling the quantification of dynamic range of expression, co-localization and co-expression of markers in the whole tissue. In this chapter, we provide the protocol for the use of the UltiMapper™ I/O PD-L1 multiplex assay, from the bench to the image analysis, as well as an overview of the current multiplex image analysis solutions. Such deep profiling could guide the development of strategies to better select immune checkpoint molecules and a better stratification of patients who will potentially benefit from immunotherapies.
