multiplex cyclic fluorescent immunohistochemical staining of cancer tissue sections

In situ immunhistochemischer Nachweis von Immunzellmarkern in Krebsgeweben

Brain metastases (BM) represent the most common central nervous system (CNS) tumors, occurring in nearly half of non-small cell lung cancer cases (NSCLC), with a poor prognosis1. An estimated 10%-20% of NSCLC patients already have BM at the time of initial diagnosis, and approximately 40% of NSCLC cases will develop BM during the course of treatment2. The tumor microenvironment (TME) is closely associated with NSCLC occurrence and BM, including various components, such as blood vessels, fibroblasts, macrophages, extracellular matrix (ECM), lymphoid, bone marrow-derived immune cells, and signaling molecules3,4. Microenvironmental immune cells play a crucial role in influencing cancer cell growth and development. Brain metastases present numerous potential treatment targets characterized by complex immunological microenvironments and signaling processes. For instance, PD-1 inhibitors have shown clinical efficacy for patients with lung cancer brain metastasis (LCBM) as an immune-checkpoint inhibitor (ICI). However, the frequency of responses to PD-1 therapy varies between primary NSCLC and LCBM5, suggesting that the tumor immune microenvironment acts as a critical ICI regulator.
Immunohistochemistry (IHC) is an invaluable tool in the fields of biology, foundation medicine, and pathology6. This detection method visualizes antigen expression through the interaction of antigen-antibody on a tissue slide7. IHC is used for diagnosing predictive markers, evaluating prognostic markers, guiding targeted therapies, and exploring the biological functions of tumor cells8. However, the traditional IHC method can only detect one biomarker at a time. To address this limitation, the innovation of immunohistochemical technology has led to the development of multiplex fluorescence immunohistochemistry (mfIHC), which allows for the simultaneous identification of multiple protein markers on the same tissue slide, both in bright field and fluorescent field9. This advancement provides accurate analysis of cell composition and molecular interactions among stromal cells, immune cells, and cancer cells within the TME.
In this study, we present a protocol for multiplex cyclic immunohistochemistry to analyze the spatial distribution of immune cells. Two primary antibodies of different species, such as rabbit and rat, are chosen for incubation simultaneously, followed by fluorescence-labeled secondary antibodies. Antigen retrieval is performed after each round of antigen-antibody reaction. Autofluorescence is blocked, and 4', 6-diamidino-2-phenylindole (DAPI) is used for staining the nuclei. The panel includes sequential detection of CD3, CD8, CD20, and CK, cells are categorized according to the markers: tumor cells (CK+), mature T cells (CD3+), cytotoxic T cells (CD3+CD8+), B cells (CD20+)10,11.

Autor im Rampenlicht: Effizienter Nachweis von Immunzellinfiltration in Krebsgeweben mittels fluoreszierender Immunhistochemie

Zyklische Multiplex-Fluoreszenz-Immunhistochemie

The challenge for immunohistochemistry is to choose the species of primary and secondary monoclonal antibodies to avoid cross-reactivity and obtain better specificity and repeatability. Gender decision is necessary to optimize the antibody dilution ratio. Meanwhile, another challenge is to align the localization of and the sub structures.The use of this protocol meet the technical evolution of the distribution of three lymphocytic subpopulations, such as CT three, positive CT three, and CT eight positive and CT 20 positive based on in cell counts and the protein expression in conserv tissues, the level of infiltration can be observed too. In our protocol, the parameter Antibodies and the Fluor secondary antibodies derive from different species at the iso mixture making us a efficient. This measure also shows a specific measure between antibody and antigen.

Multiplex-zyklische fluoreszierende immunhistochemische Färbung von Krebsgewebeschnitten

Besorgen Sie sich zunächst zuvor vorbereitete Objektträger mit Krebsgewebeschnitten. Bereiten Sie eine Arbeitsmischung aus Primärantikörpern vor. Geben Sie den Antikörperkomplex auf den Objektträger und inkubieren Sie ihn eine Stunde lang bei Raumtemperatur.Waschen Sie den Objektträger dann dreimal fünf Minuten lang mit 0,1 % Tween PBS. Bereiten Sie eine fluoreszierende Sekundärantikörpermischung vor. Geben Sie es tropfenweise auf den Objektträger und inkubieren Sie es eine Stunde lang bei Raumtemperatur.Führen Sie dann eine zweite Runde der Primärantikörperinkubation bei Raumtemperatur für eine Stunde durch. Waschen Sie den Objektträger dreimal mit 0,1 % Tween PBS für jeweils fünf Minuten. Entfernen Sie mit einem Filterpapier überschüssiges Wasser aus dem Umfang des Gewebes.Führen Sie in ähnlicher Weise eine zweite Runde der Sekundärantikörperinkubation durch und waschen Sie, wie zuvor gezeigt. Fügen Sie nun dem Gewebe Kaliumpermanganat-Reagenz hinzu. Spülen Sie es nach einer Minute fünf Minuten lang mit fließendem Wasser ab.Führen Sie eine Dehydrierung mit steigenden Alkoholkonzentrationen durch. Fügen Sie schließlich ein DAPI hinzu, um den Gewebeschnitt vollständig abzudecken, und legen Sie ein Deckglas für die multispektrale Bildgebung an. Die Multiplex-Immunfluoreszenzbildgebung zeigte das Vorhandensein von CD-3-positiven, CD-8-positiven, CD-20-positiven Zellen im Lungenkrebs-Hirnmetastasengewebe.

multiplex-cyclic-fluorescent-immunohistochemical-staining-cancer

Research

JoVE Journal

Cancer Research

Multiplex Cyclic Fluorescent Immunohistochemical Staining of Cancer Tissue Sections

56 Aufrufe.  The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital. Besorgen Sie sich zunächst zuvor vorbereitete Objektträger mit Krebsgewebeschnitten. Bereiten Sie eine Arbeitsmischung aus Primärantikörpern vor. Geben Sie den Antikörperkomplex auf den Objektträger und inkubieren Sie ihn eine Stunde lang bei Raumtemperatur.Waschen Sie den Objektträger dann dreimal fünf Minuten lang mit 0,1 % Tween PBS. Bereiten Sie eine fluoreszierende Sekundärantikörpermischung vor. Geben Sie es tropfenweise auf den Objektträger und inkubieren Sie es eine ...

Serie: Multiplex-zyklische fluoreszierende immunhistochemische Färbung von Krebsgewebeschnitten

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.