Literature Analysis: Application of Multiplex Fluorescence IHC in Deciphering Metastatic Mechanisms of Colorectal Cancer

1.Literature Information

Research Focus: Spatiotemporal evolution of tumor metastases under immune selection, exploring how the immune microenvironment influences colorectal cancer (CRC) evolution in different metastatic organs, and establishing an immune evaluation system for tumor metastasis

Key Technology: Multiplex Fluorescence Immunohistochemistry (mIHC), Genomics, Immunomics

Sample Information: Biological specimens from two CRC patients (P210 and P45), including primary tumors and metastases from multiple organs (lung, liver, peritoneum); multi-regional resection specimens from tumor tissues and surrounding areas

Key Product Utilized: Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD.

2. Research Background

Colorectal cancer (CRC) is one of the most common malignant tumors globally, and metastasis is the major cause of treatment failure and poor prognosis in CRC patients. The metastatic process of CRC is a complex and dynamic biological process involving interactions between tumor cells and the immune microenvironment. Immune selection plays a crucial role in regulating tumor clonal evolution and metastatic progression, but the underlying mechanisms by which the immune microenvironment modulates CRC metastasis in different organs remain incompletely understood.

Traditional research techniques such as genomics and single-color immunohistochemistry are limited in comprehensively analyzing the spatial interactions between tumor cells and immune cells, as well as the dynamic changes of the immune microenvironment during metastasis. Multiplex Fluorescence Immunohistochemistry (mIHC) technology enables simultaneous detection of multiple molecular markers on a single tissue section, providing a powerful tool for visualizing the spatial distribution of immune cells and tumor cells, quantifying immune infiltration, and deciphering the complex crosstalk between tumor and immune systems. This technology has become increasingly important in exploring tumor metastatic mechanisms and optimizing immunotherapeutic strategies.

3. Research Approach

To investigate the spatiotemporal evolution of CRC metastases under immune selection, the research team collected biological specimens from two CRC patients (P210 and P45), including primary tumors and metastases from multiple organs (lung, liver, peritoneum). Multi-regional resection specimens were obtained from large tumor tissues and their surrounding areas to ensure comprehensive coverage of tumor heterogeneity.

The team employed the Multiplex Fluorescence IHC Staining Kit (abs50032) from ANT BIO PTE. LTD. to perform mIHC staining. A panel of 7 indicators was used, with a maximum of 4 indicators and 5 colors detected on a single slide. The detected markers included immune cell markers (CD3, CD8, CD20, FoxP3), proliferation marker (Ki67), tumor cell marker (CK), and immune checkpoint molecule (PD-L1).

Three metrics were used to quantify immune infiltration: whole-slide immune cell density (WS), hot spot density (HSs, the three regions with the highest infiltration density), and immune score based on CD3+/CD8+ T cell density in the tumor center (CT) and invasive margin (IM). Additionally, spatial proximity analysis between Ki67+ tumor cells and CD3+ cells was conducted to explore the interaction between tumor proliferation and immune cells. Genomic and immunomic analyses were combined to comprehensively decipher immune escape mechanisms and clonal evolution patterns during CRC metastasis.

4. Research Results

Firstly, the study revealed that the clonal evolution pattern during CRC metastasis depends on the immune microenvironment of the metastatic site. Under immune selection pressure, tumor cells undergo immunoediting, leading to the emergence of immune-escape clones, which are crucial for metastatic progression.

Secondly, the mIHC staining results using ANT BIO PTE. LTD.'s abs50032 kit successfully visualized the spatial distribution of immune cells and tumor cells in the CRC microenvironment. Spatial proximity analysis between Ki67+ tumor cells (CK+Ki67+) and CD3+ T cells uncovered new genetic evidence of neoantigen loss. The analysis of three groups (LoNo, HiNo, HiYes) showed that the shortest distance between T cells and tumor cells gradually decreased, and the percentage of CD3+ T cells adjacent to both proliferative and non-proliferative tumor cells in the CT increased significantly, with statistically significant differences between groups.

Thirdly, the research established an immune evaluation system for tumor metastasis, revealing the relationship between recurrence risk and immune score, immunoediting, and tumor burden. A higher immune score was associated with a lower recurrence risk, while enhanced immunoediting and increased tumor burden were positively correlated with poor prognosis.

Finally, the team proposed a parallel selection model explaining CRC metastatic progression, where branches can be traced back to immune-escape clones. This model provides new insights into tumor dissemination and the development of immunotherapeutic strategies.

5. Product Empowerment

The Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD. was a core technical support for this research. This kit enabled simultaneous detection of multiple key markers (CD3, CD8, CD20, FoxP3, Ki67, CK, PD-L1) on a single tissue section, realizing 4-indicator 5-color staining on a single slide, which was essential for visualizing the spatial distribution of immune cells and tumor cells and analyzing their interactions.

The abs50032 kit possesses distinct advantages that guaranteed the success of the study: 1) It supports up to 6 indicators with 7 colors (including DAPI) of multiplex labeling, meeting the requirement of multi-marker simultaneous detection in complex tumor microenvironment research; 2) The optimized SuperNova series dyes exhibit higher fluorescence intensity and longer signal duration compared to conventional dyes, ensuring clear and stable imaging results for subsequent quantitative analysis; 3) The kit is user-friendly, requiring only basic IHC experience for operation without the need for specialized equipment, and is accompanied by a detailed protocol for standardized experimentation; 4) ANT BIO PTE. LTD. provides comprehensive mIHC services covering the entire process from pre-experiment verification, staining, and imaging to data analysis, including single-cell quantitative analysis (percentage and density of single-positive and double-positive cells), tissue area calculation, and specific pathological morphology area measurement, which greatly facilitated the smooth implementation of the research.

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.

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work in document 1226.docx (Application of mIHC in Colorectal Cancer Metastatic Mechanisms). All intellectual property (e.g., images, data) of the original publication shall belong to the corresponding journal and research team. For any infringement, please contact us promptly and we will take immediate action.

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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.