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J Transl Med. 2017 Oct 12;15(1):206. doi: 10.1186/s12967-017-1311-8.

Understanding immune phenotypes in human gastric disease tissues by multiplexed immunohistochemistry.

Ying L1,2, Yan F1,3, Meng Q3, Yuan X1, Yu L4, Williams BRG5, Chan DW6, Shi L7, Tu Y8, Ni P1, Wang X9, Xu D10,11,12, Hu Y13.

Author information

1
Faculty of Medical Laboratory Science, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 227 Chongqing Road South, Shanghai, 200025, China.
2
Department of Tea Science, Zhejiang University, Hangzhou, China.
3
Institute of Ageing Research, Hangzhou Normal University School of Medicine, Hangzhou, China.
4
Department of General Surgery, Shanghai Jiao Tong University Affiliated First People's Hospital, Shanghai, China.
5
Hudson Institute of Medical Research, Department of Molecular and Translational Science, Monash University, Clayton, VIC, Australia.
6
Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P. R. China.
7
Department of Microbiology and Immunology, Nanjing University of Chinese Medicine, Nanjing, 210046, China.
8
Cell Signaling Technology, Inc., Asia Pacific, Danvers, USA.
9
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
10
Faculty of Medical Laboratory Science, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 227 Chongqing Road South, Shanghai, 200025, China. dakang.xu@monash.edu.
11
Institute of Ageing Research, Hangzhou Normal University School of Medicine, Hangzhou, China. dakang.xu@monash.edu.
12
Hudson Institute of Medical Research, Department of Molecular and Translational Science, Monash University, Clayton, VIC, Australia. dakang.xu@monash.edu.
13
Faculty of Medical Laboratory Science, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 227 Chongqing Road South, Shanghai, 200025, China. ichunhu@126.com.

Abstract

BACKGROUND:

Understanding immune phenotypes and human gastric disease in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses.

METHODS:

We developed a novel 4-color mIHC assay based on tyramide signal amplification that allowed us to reliably interrogate immunologic checkpoints, including programmed death-ligand 1 (PD-L1), cytotoxic T cells (CD8+T) and regulatory T cells (Foxp3), in formalin-fixed, paraffin-embedded tissues of various human gastric diseases. By observing cell phenotypes within the disease tissue microenvironment, we were able to determine specific co-localized staining combinations and various measures of cell density.

RESULTS:

We found that PD-L1 was expressed in gastric ulcer and in tumor cells (TCs), as well as in tumor-infiltrating immune cells (TIICs), but not in normal gastric mucosa or other gastric intraepithelial neoplastic tissues. Furthermore, we found no significant reduction in CD8+T cells, whereas the ratio of CD8+T:Foxp3 cells and CD8+T:PD-L1 cells was suppressed in tumor tissues and elevated in adjacent normal tissues. An unsupervised hierarchical analysis also identified correlations between CD8+T and Foxp3+ tumor-infiltrating lymphocyte (TIL) densities and average PD-L1 levels. Three main groups were identified based on the results of CD8+T:PD-L1 ratios in gastric tumor tissues. Furthermore, integrating CD8+T:Foxp3 ratios, which increased the complexity for immune phenotype status, revealed 6-7 clusters that enabled the separation of gastric cancer patients at the same clinical stage into different risk-group subsets.

CONCLUSIONS:

Characterizing immune phenotypes in human gastric disease tissues via multiplexed immunohistochemistry may help guide PD-L1 clinical therapy. Observing unique disease tissue microenvironments can improve our understanding of immune phenotypes and cell interactions within these microenvironments, providing the ability to predict safe responses to immunotherapies.

KEYWORDS:

Human gastric disease; Immune phenotypes; Multiplexed immunohistochemistry

PMID:
29025424
PMCID:
PMC5639762
DOI:
10.1186/s12967-017-1311-8
[Indexed for MEDLINE]
Free PMC Article

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