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Cancer Res Treat. 2019 May 27. doi: 10.4143/crt.2019.195. [Epub ahead of print]

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

Koh J1, Kwak Y1,2, Kim J3, Kim WH1,3,4.

Author information

Department of Pathology, Seoul National University Hospital, Seoul, Korea.
Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea.
Cancer Research Institute, Seoul National University, Seoul, Korea.
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.



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.


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.


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.


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

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