Comprehensive Solution: 7-Color Multiplex Fluorescence IHC for Thyroid Tumor Immune Microenvironment Analysis

1. Introduction to Tumor Immune Microenvironment and Multiplex IHC Technology

A developed tumor is a complex tissue composed not only of tumor cells but also stromal cells, inflammatory cells, vascular systems, and extracellular matrix (ECM). The sum of these components is defined as the Tumor Microenvironment (TME), which plays a crucial role in tumor malignant progression, immune escape, and therapeutic resistance [1]. Developing safe and effective tumor immunotherapies based on the characteristics of TME is one of the most promising hotspots in the field of tumor treatment currently [2]. To date, multiple approved drugs have been used for the treatment of different types of tumors, achieving remarkable achievements in tumor therapy [2, 3]. However, tumor cells can also negatively regulate the body's immune response through various pathways, thereby escaping immune surveillance.

Therefore, immunotherapy still faces enormous challenges, such as unpredictable therapeutic effects and patient responses, tumor immunotherapy resistance, and the lack of effective biomarkers [4]. The positive response to immunotherapy usually depends on the interaction between tumor cells and immune regulation within the TME. Under these interactions, TME plays an important role in inhibiting or enhancing immune responses. Understanding the interaction between immunotherapy and TME is not only the key to analyzing the mechanism of action but also of great significance for providing new methods to improve the efficacy of current immunotherapy

Multiplex immunohistochemistry (mIHC) technology has unique advantages in evaluating the expression abundance, interaction, and spatial phenotypic information of various cellular components in the TME. It can simultaneously label multiple targets on the same section, helping researchers quantitatively evaluate cell phenotypes and activity, comprehensively depict the tumor immune microenvironment and in-situ spatial information between cells. This enables us to gain a deeper understanding of the mechanism of tumorigenesis and is more likely to predict tumor response to treatment [6-9]. In this solution, we used TissueFAXS Cytometry panoramic tissue flow cytometry quantitative analysis technology from TissueGnostics combined with the 7-Color Multiplex Fluorescence IHC Staining Kit (abs50015) from the Absin product line of ANT BIO PTE. LTD. to carry out multi-color immunohistochemistry application exploration in tonsil tumor samples.

2. Panel and Dye Information

Panel and dye information (multi-color labeling of tumor cells and different immune cell phenotypes in samples): On paraffin-embedded thyroid cancer tissue sections, TSA tyramide signal amplification technology was used. HRP-labeled secondary antibodies were adopted, and HRP catalyzed the luciferin substrate added to the system to generate activated luciferin substrate. The activated substrate could covalently bind to tyrosine on the antigen, enabling stable covalent binding of luciferin on the sample.

Subsequently, non-covalently bound antibodies were washed away by heat repair to avoid cross-reaction, and multiple fluorescence labeling was achieved through repeated cycles. After simultaneously labeling 6 biomarkers using the new generation TSA staining technology of the 7-Color Multiplex Fluorescence IHC Staining Kit from ANT BIO PTE. LTD., continuous full-spectrum high-magnification imaging was performed using the TissueFAXS Spectra system to construct panoramic (multi-spectral) virtual sections. Clear multi-color sample images and independent signals of each single channel were obtained, depicting the complex tumor immune microenvironment from a multi-target perspective.

3. Acquisition and Spectral Unmixing of Multi-Color Immunofluorescence Images

Multiplex IHC staining spectral unmixing of human thyroid cancer tissue. Independent true staining images of each antibody with background autofluorescence, autofluorescence of blood cells/collagen, etc., and non-specific staining of mucus clumps removed. 7-color multispectral images and spectral unmixing results of human thyroid cancer FFPE.

4. In-Situ Protein Cell Subset Analysis of Tissues

The patented nuclear recognition algorithm unique to TG provides a precise single-cell recognition basis in tissue in-situ, and the visual forward and reverse backtracking verification technology of big data further guarantees its accuracy. The unique tissue in-situ flow analysis function of TG screens target cells layer by layer, which can obtain quantitative expression and spatial localization information of double-labeled, triple-labeled and even more labeled cells. It can personally analyze the phenotypic characteristics of any cell in tissue sections, providing strong support for in-situ visual precise typing of cell subsets.

Multi-color labeling using ANT BIO PTE. LTD.'s 7-Color Multiplex Fluorescence IHC technology and immune cell typing screening and single-target quantification of samples using TissueFAXS Cytometry technology. The figure shows the layer-by-layer screening and identification of CD20+CD4-CD3- cells through the unique tissue flow scatter plot (green circle in the left figure). The scatter plot can be traced back to each single cell in the image. First, the CD20+ positive cell population (upper right figure) sets a Cutoff line for this cell population to screen positive cells. And this positive cell population is quoted to the next scatter plot to screen CD4 and CD3 negative cell populations (lower right figure). Figure 3. Schematic diagram of visual forward and reverse backtracking verification. Each indicator identified in the image can be corresponding one by one in the scatter plot on the right, and each point in the scatter plot can be accurately traced back to the image for easy observation.

5. Spatial Multi-Dimensional Quantitative Analysis of Tumor Microenvironment

Using TissueFAXS Cytometry technology, multi-modal and cross-scale spatial relationship quantitative analysis research from single cells, cell populations, tissue structures to organ levels can be realized. For example, in the research direction of tumor microenvironment, spatial data mining can be extended to the tumor sub-microenvironment, further refining the complex interaction relationships between various labeled cells and single cells or cell populations, between single cells and tumor tissues, between specific cell populations and tumor tissues, and between tumor tissues and blood vessels in the tumor sub-microenvironment.

Furthermore, more abundant sociological relationships can be derived, and scientific information with cell communities as functional units can be mined, providing an important data source for explaining the occurrence and development of diseases. Spatial distribution map of cell populations centered on tumors. Using the artificial intelligence Classifier function in TissueFAXS Cytometry technology to specifically identify tumor and stromal regions of tissue samples, and centering on the tumor region, single-cell quantitative analysis of the spatial distribution of immune cells in the microenvironment with micron-level precision. Different colors represent different distance ranges from the tumor. The number of immune cell infiltrations at different distances from the tumor is indicated by the number of points of different colors in the scatter plot on the right.

Schematic diagram of the analysis of the interaction cell social relationship between tumor cells (green) and NK cells (yellow). The distance relationship between tumor cells and NK cells was analyzed by TissueFAXS Cytometry technology, NK cells within 30 μm around tumor cells were counted, and indicated by connecting lines.

6. TissueFAXS Cytometry Panoramic Tissue Flow Cytometry Quantitative Analysis Platform

Based on the precise single-cell quantification in tissue in-situ, the powerful artificial intelligence (AI) characteristic tissue recognition, and the pixel-level fine spatial quantitative analysis, the powerful arithmetic processing functions are freely interactively combined to provide a one-stop solution from staining to imaging to data mining. It can achieve precise customized analysis in different complex microenvironments such as tumor immunology and neurobiology.

Figure 6. Schematic diagram of the TissueFAXS Cytometry workflow. Tissue sections (paraffin/frozen) → ≥10-color labeling → TissueFAXS Spectra fully automatic multispectral imaging system → Panoramic virtual sections → StrataQuest App full-dimensional big data in-depth mining.

7. References

1. Balkwill, F.R., M. Capasso, and T. Hagemann, The tumor microenvironment at a glance. J Cell Sci, 2012. 125(Pt 23): p. 5591-6.
2. Bejarano, L., M.J.C. Jordao, and J.A. Joyce, Therapeutic Targeting of the Tumor Microenvironment. Cancer Discov, 2021. 11(4): p. 933-959.
3. Galon, J. and D. Bruni, Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov, 2019. 18(3): p. 197-218.
4. Hegde, P.S. and D.S. Chen, Top 10 Challenges in Cancer Immunotherapy. Immunity, 2020. 52(1): p. 17-35.
5. Tang, H., J. Qiao, and Y.X. Fu, Immunotherapy and tumor microenvironment. Cancer Lett, 2016. 370(1): p. 85-90.
6. Tsujikawa, T., et al., Quantitative Multiplex Immunohistochemistry Reveals Myeloid-Inflamed Tumor-Immune Complexity Associated with Poor Prognosis. Cell Rep, 2017. 19(1): p. 203-217.
7. Chen, S., et al., Multiomic Analysis Reveals Comprehensive Tumor Heterogeneity and Distinct Immune Subtypes in Multifocal Intrahepatic Cholangiocarcinoma. Clin Cancer Res, 2022. 28(9): p. 1896-1910.
8. Chen, X., et al., CD8(+) T effector and immune checkpoint signatures predict prognosis and responsiveness to immunotherapy in bladder cancer. Oncogene, 2021. 40(43): p. 6223-6234.
9. Wang, T., et al., Spatial distribution and functional analysis define the action pathway of Tim-3/Tim-3 ligands in tumor development. Mol Ther, 2022. 30(3): p. 1135-1148.

8. High-Quality Multiplex Fluorescence IHC Kits Provided by ANT BIO PTE. LTD.

The Multiplex Fluorescence IHC Kits from the Absin product line of ANT BIO PTE. LTD. can achieve multi-labeling on a single slide. Whether it is observing the tumor microenvironment, immune infiltration, performing cell counting or co-localization analysis, we can achieve it. Currently, the maximum can reach six indicators and seven colors~

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10. Disclaimer

This article is AI-compiled and interpreted based on the original work in document 1225.docx (7-Color Thyroid Tissue Tumor Immune Microenvironment Solution). All intellectual property (e.g., images, data) of the original publication shall belong to the relevant research team and institutions. For any infringement, please contact us promptly and we will take immediate action.

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