Literature Analysis: Application of Multiplex Fluorescence IHC in Characterizing Subtypes and Functions of T Cells in Colorectal Cancer

1. Literature Information
Journal: Nature
DOI: 10.1038/s41586-018-0694-x
Published Online: Accelerated Article Preview
Authors: Lei Zhang, Xin Yu, Liangtao Zheng, Yuanyuan Zhang, Yansen Li, et al.
Research Focus: Exploring the development and migration characteristics of T cells in tumors by tracking T cell receptor (TCR) lineage, establishing the STARTRAC index to analyze dynamic relationships among different T cell subsets in colorectal cancer (CRC), and characterizing the subtypes and functions of T cells using multiplex fluorescence IHC
Key Technology: Single-cell RNA sequencing, flow cytometry, Multiplex Fluorescence Immunohistochemistry (mIHC)
Key Product Utilized: Multiplex Fluorescence IHC Staining Kits and related services from the Absin product line of ANT BIO PTE. LTD.

2. Research Background
T cells are key elements in cancer immunotherapy, playing a crucial role in anti-tumor immune responses. However, certain fundamental characteristics of T cells, such as their development and migration within tumors, remain elusive. The T cell receptor (TCR), essential for recognizing foreign and self-antigens, can serve as a lineage marker to track T cells in tumors. Deciphering the dynamic relationships and functional characteristics of different T cell subsets in CRC is of great significance for optimizing immunotherapeutic strategies and improving treatment efficacy.

 
Traditional research techniques such as single-cell sequencing and flow cytometry can provide information on T cell subsets and their transcriptomic profiles but fail to visualize the spatial distribution and in situ interactions of T cells in tumor tissues. Multiplex Fluorescence Immunohistochemistry (mIHC) technology enables simultaneous detection of multiple T cell markers on a single tissue section, realizing in situ localization and functional analysis of T cell subsets. This technology complements sequencing and flow cytometry techniques, providing a comprehensive understanding of T cell biology in the tumor microenvironment.

 

 
3. Research Approach
To explore the characteristics of T cell subsets in CRC, the research team obtained transcriptomes of 11,138 single T cells from 12 CRC patients and established the STARTRAC (Single T-cell Analysis by RNA-sequencing and TCR Tracking) index. This index was used to quantitatively analyze the dynamic relationships among 20 functionally and clonally distinct T cell subsets.

 

To verify the presence of T cell subsets and analyze their in situ expression patterns, the team employed multiplex fluorescence IHC technology. They performed multi-color IHC staining using markers such as CD3, CD4, CD8, and FOXP3 to identify T cell subsets in CRC tissues.

Additionally, co-expression analysis of Ki-67, CD8, PD-1, and TIM-3 was conducted to characterize the functional state of exhausted CD8+ T cells (TEX). Co-expression of CD3, FOXP3, and RORγ was also detected to explore specific T cell subsets. All mIHC experiments were supported by ANT BIO PTE. LTD.'s Absin Multiplex Fluorescence IHC Staining Kits and related technical services, covering the entire process from pre-experiment verification, staining, imaging to data analysis.

4. Research Results
Firstly, the STARTRAC index analysis revealed various functional, migratory, and developmental connections among different T cell subsets in CRC. The data, combined with previous findings in mouse models, uncovered a TCR-dependent trajectory of tumor-infiltrating CD8+ TEM cells to TEMRA and TEX cells, suggesting therapeutic strategies to promote the conversion of TEX to TEMRA.

Secondly, mIHC staining results confirmed the presence of multiple T cell subsets in CRC tissues. Representative staining examples showed co-expression of Ki-67, CD8, PD-1, and TIM-3 in CD8+ TEX cells, indicating the exhausted state of these cells. Additionally, co-expression of CD3, FOXP3, and RORγ was observed (marked by white arrows in staining images), identifying specific T cell subsets with unique phenotypic characteristics.

Thirdly, in microsatellite instability (MSI) patients, enrichment of CXCL13+BHLHE40+IFNG+ TH1-like cells was found. This finding not only provides a theoretical basis for the high response rate of these patients to checkpoint blockade therapy but also inspires considerations for cell therapy targeting these cells.

Finally, a compendium dataset of differentially expressed genes (such as IGFLR1) was established and made available through the interactive portal http://crc.cancer-pku.cn, serving as a resource for further exploring T cells, identifying new regulatory mechanisms, and discovering therapeutic targets.

5. Product Empowerment
ANT BIO PTE. LTD.'s Absin Multiplex Fluorescence IHC Staining Kits and technical services provided critical support for this research. The Absin Multiplex Fluorescence IHC service covers the entire process from pre-experiment verification, staining, imaging to data analysis, supporting up to 6 indicators with 7 colors (including DAPI) for multi-color IHC staining (up to 4 indicators with 5 colors for tissue microarrays). The service is applicable to various sample types, including paraffin sections, frozen sections, and tissue microarrays (TMA), meeting the diverse sample requirements of the research.

The Absin Multiplex Fluorescence IHC Staining Kits consist of high-quality components such as TSA fluorescent dyes, signal amplification reaction solution, mouse/rabbit universal or anti-rabbit HRP-labeled secondary antibodies, anti-fluorescence quenching mounting medium, and DAPI. These components ensure high signal intensity, stable fluorescence, and clear imaging results, enabling accurate detection of co-expression patterns of multiple T cell markers. The technical services provided by ANT BIO PTE. LTD. include single-cell analysis results of user-specified regions, such as the percentage and density of single-positive and double-positive cells relative to nucleated cells, calculation of tissue area and specific pathological morphology area, and quantitative results for each core of TMA. These comprehensive services and high-quality products ensured the reliability and accuracy of the mIHC experimental results, facilitating the in-depth exploration of T cell subsets in CRC.

6. Brand Mission
ANT BIO PTE. LTD. is dedicated to empowering life science research through the provision of high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) are strategically positioned to cover a full spectrum of research needs: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. We strive to support researchers in unlocking scientific mysteries, advancing medical progress, and addressing unmet clinical needs through continuous innovation and customer-centric services.

7. Related Product List

Catalog No.	Product Name	Specification
abs50086	Two-Color Multiplex Immunofluorescence IHC Staining Kit (Anti-Rabbit Secondary Antibody)	100T
abs50087	Two-Color Multiplex Immunofluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody)	100T
abs50088	Three-Color Multiplex Immunofluorescence IHC Staining Kit (Anti-Rabbit Secondary Antibody)	100T
abs50089	Three-Color Multiplex Immunofluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody)	100T
abs50012	Four-Color Multiplex Immunofluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50168	Four-Color Multiplex Immunofluorescence IHC Staining Kit B (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50013	Five-Color Multiplex Immunofluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50029	Five-Color Multiplex Immunofluorescence IHC Staining Kit (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50030	Six-Color Multiplex Immunofluorescence IHC Staining Kit (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50048	Six-Color Multiplex Immunofluorescence IHC Staining Kit (Plus) (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50049	Six-Color Multiplex Immunofluorescence IHC Staining Kit (Plus) (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50015	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50031	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50037	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (Plus) (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50038	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (Plus) (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50165	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (770 Dye Enhanced Version) (Anti-Rabbit Secondary Antibody)	20T/50T/100T
abs50166	Seven-Color Multiplex Immunofluorescence IHC Staining Kit (770 Dye Enhanced Version) (Mouse/Rabbit Universal Secondary Antibody)	20T/50T/100T
abs50018	Ten-Color Multiplex Immunofluorescence IHC Staining Kit	100T
abs50083	Lung Cancer Tumor Microenvironment Multiplex Immunofluorescence IHC Detection Kit (I)	20T
abs50084	Lung Cancer Tumor Microenvironment Multiplex Immunofluorescence IHC Detection Kit (II)	20T

8. Disclaimer
This article is AI-compiled and interpreted based on the original work in document 1226.docx (Application of mIHC in Exploring the Characterization and Function of Different T Cell Subtypes) and the original literature DOI: 10.1038/s41586-018-0694-x. All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

9. Brand Promotion Copy
ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs
At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.