Cancer Res Treat.  2020 Jan;52(1):98-108. 10.4143/crt.2019.195.

High-Throughput Multiplex Immunohistochemical Imaging of the Tumor and Its Microenvironment

• Koh J
• Kwak Y
• Kim J
• Kim WH

• KMID: 2501205
• DOI: http://doi.org/10.4143/crt.2019.195

Abstract

Purpose
The aim of this study was to develop a formalin-fixed paraffin-embedded (FFPE) tissue based multiplex immunochemistry (mIHC) method for high-throughput comprehensive tissue imaging and demonstrate its feasibility, validity, and usefulness.
Materials and Methods
The mIHC protocol was developed and tested on tissue microarray slides made from archived gastric cancer (GC) tissue samples. On a single FFPE slide, cyclic immunochemistry for multiple markers of immune cells and cytokeratin for tumor cells was performed; hematoxylin staining was used for demarcation of nuclei. Whole slides were digitally scanned after each cycle. For interpretation of mIHC results, we performed computer-assisted image analysis using publicly available software.
Results
Using mIHC, we were able to characterize the tumor microenvironment (TME) of GCs with accurate visualization of various immune cells harboring complex immunophenotypes. Spatial information regarding intratumoral and peritumoral TME could be demonstrated by digital segmentation of image guided by cytokeratin staining results. We further extended the application of mIHC by showing that subcellular localization of molecules can be achieved by image analysis of mIHC results.
Conclusion
We developed a robust method for high-throughput multiplex imaging of FFPE tissue slides. The feasibility and adaptability of mIHC suggest that it is an efficient method for in situ single-cell characterization and analysis.

Keyword

Immunohistochemistry; Computer-assisted image processing; Tissue microarray analysis; Tumor microenvironment

Figure

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  Fig. 1. Overview of multiplex immunohistochemistry (IHC) process. After preprocessing, hematoxylin staining for identification of nuclei is the first step, followed by cyclic IHC including steps of antigen retrieval, antibody incubation, whole slide scanning, and antibody stripping. This cyclic IHC can be repeated up to 10 times and the panel of IHC markers can be customized. FFPE, formalinfixed paraffin-embedded.

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  Fig. 2. Post-processing of cyclic immunohistochemistry (IHC) images for further analysis. (A) Bright-field images of cyclic IHC on the same slide should be aligned accurately for correct analysis. (B) Determination of cut-off value for each staining can be digitally guided for optimization. Scale bars=125 μm

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  Fig. 3. Intuitive visualization and characterization of tumor microenvironment by multiplex immunohistochemistry. (A) Representative images of different markers in the same area are shown. Distribution of T-cell subsets (B) and immune cells expressing inhibitory molecules (C) can be visualized after assigning pseudocolors. The area of the images is 1/64 of a single core. Scale bars=50 μm.

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  Fig. 4. In-depth computational analysis of tumor microenvironment using metadata from multiplex immunohistochemistry. Number/density of cells with a certain immunophenotype can be directly compared (A) and comprehensive analysis, including unsupervised clustering can be performed (B). EBV, Epstein-Barr virus; MSI-H, microsatellite instability–low.

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  Fig. 5. Spatial information of tumor microenvironment retrieved using multiplex immunohistochemistry. (A) Spatial relationship between tumor cells and tumor-infiltrating immune cells can be assessed by digitally analyzing the distance between cytokeratin (CK)-positive tumor cells and target immune cells of interest. Scale bars=100 μm. (B) Using the viSNE algorithm, various signatures representing each type of immune cell can be identified, and immune cell clusters based on the combination of immunostained markers can be spatially segmented into distinct subgroups. MSS, microsatellite stable; MSI, microsatellite instability.

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  Fig. 6. Subcellular localization of molecules using multiplex immunohistochemistry. Immunostaining of H3K27me3 and EZH2 was performed on the same slide (A) and the expression level of H3K27me3 is depicted according to the expression and subcellular location of EZH2 (B). Scale bars=50 μm (A), 20 μm (insets in A).

Reference

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