multiplex cyclic fluorescent immunohistochemical staining of cancer tissue sections

Rilevamento immunoistochimico in situ di marcatori di cellule immunitarie nei tessuti tumorali

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.

Autore in primo piano: Rilevamento efficiente dell'infiltrazione di cellule immunitarie nei tessuti tumorali mediante immunoistochimica fluorescente

Immunoistochimica fluorescente ciclica multiplex

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.

Colorazione immunoistochimica fluorescente ciclica multiplex di sezioni di tessuto tumorale

Per iniziare, procurati dei vetrini precedentemente preparati contenenti sezioni di tessuto tumorale. Preparare una miscela di lavoro di anticorpi primari. Aggiungere il complesso anticorpale sulla sezione del vetrino e incubare per un'ora a temperatura ambiente.Quindi lavare il vetrino tre volte con PBS allo 0,1% per cinque minuti. Preparare una miscela di anticorpi secondari fluorescenti. Aggiungilo goccia a goccia sul vetrino e incubalo a temperatura ambiente per un'ora.Quindi eseguire un secondo ciclo di incubazione degli anticorpi primari a temperatura ambiente per un'ora. Lavare il vetrino tre volte con PBS allo 0,1% per cinque minuti ciascuna. E usando una carta da filtro, rimuovi l'acqua in eccesso dal perimetro del tessuto.Allo stesso modo, eseguire un secondo ciclo di incubazione e lavaggio secondario degli anticorpi come dimostrato in precedenza. Ora, aggiungi il reagente del permanganato di potassio al tessuto. Dopo un minuto, sciacquare con acqua corrente per cinque minuti.Eseguire la disidratazione con concentrazioni crescenti di alcol. Infine, aggiungere un DAPI per coprire completamente la sezione di tessuto e posizionare un vetrino coprioggetto per l'imaging multispettrale. L'imaging a immunofluorescenza multiplex ha indicato la presenza di cellule CD tre positive, CD otto positive, CD 20 positive nel tessuto delle metastasi cerebrali del cancro del polmone.

multiplex-cyclic-fluorescent-immunohistochemical-staining-cancer

Research

JoVE Journal

Cancer Research

Multiplex Cyclic Fluorescent Immunohistochemical Staining of Cancer Tissue Sections

56 Views.  The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital. Per iniziare, procurati dei vetrini precedentemente preparati contenenti sezioni di tessuto tumorale. Preparare una miscela di lavoro di anticorpi primari. Aggiungere il complesso anticorpale sulla sezione del vetrino e incubare per un'ora a temperatura ambiente.Quindi lavare il vetrino tre volte con PBS allo 0,1% per cinque minuti. Preparare una miscela di anticorpi secondari fluorescenti. Aggiungilo goccia a goccia sul vetrino e incubalo a temperatura ambiente per un'ora.Quind...

Video: Colorazione immunoistochimica fluorescente ciclica multiplex di sezioni di tessuto tumorale

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.