Abstract
The tumor microenvironment (TME), composed of immune cells, antigens, and local soluble factors, is integral to cancer development and progression. Traditional techniques such as immunohistochemistry, immunofluorescence, or flow cytometry limit the analysis of spatial data and cellular interactions within the TME, as they are restricted to colocalization of a small number of antigens or the loss of tissue architecture. Multiplex fluorescent immunohistochemistry (mfIHC) allows for detection of multiple antigens within a single tissue sample, providing a more comprehensive description of tissue composition and spatial interactions within the TME. This technique utilizes antigen retrieval, application of primary and secondary antibodies, followed by a tyramide-based chemical reaction to covalently bind a fluorophore to an epitope of interest and, eventually, stripping of the antibodies. This allows for multiple rounds of antibody application without concern for species cross-reactivity, as well as signal amplification which abrogates the autofluorescence that frequently plagues analysis of fixed tissues. As such, mfIHC can be used to quantify multiple cellular populations and their interactions, in situ, unlocking key biologic data that was previously unavailable. This chapter provides an overview of the experimental design, staining, and imaging strategies using a manual technique in formalin-fixed paraffin-embedded tissue sections.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gout S, Huot J (2008) Role of cancer microenvironment in metastasis: focus on colon cancer. Cancer Microenviron 1(1):69–83
Chen F, Zhuang X, Lin L et al (2015) New horizons in tumor microenvironment biology: challenges and opportunities. BMC Med 13:45
Paluskievicz CM, Cao X, Abdi R, Zheng P, Liu Y, Bromberg JS (2019) T Regulatory Cells and priming the suppressive tumor microenvironment. Front Immunol 10:2453
Schulz M, Salamero-Boix A, Niesel K, Alekseeva T, Sevenich L (2019) Microenvironmental regulation of tumor progression and therapeutic response in brain metastasis. Front Immunol 10:1713
Katikireddy KR, O’Sullivan F (2011) Immunohistochemical and immunofluorescence procedures for protein analysis. Methods Mol Biol 784:155–167
Tan WCC, Nerurkar SN, Cai HY et al (2020) Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond) 40(4):135–153
Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70(1):46–58
Taylor CR, Levenson RM (2006) Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II. Histopathology 49(4):411–424
Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2(12):910–919. https://doi.org/10.1038/nmeth817
Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A (2017) Flow cytometry: basic principles and applications. Crit Rev Biotechnol 37(2):163–176
Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM (2010) New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev Vaccines 9(6):601–616
Boisson A, Noel G, Saiselet M et al (2021) Fluorescent multiplex immunohistochemistry coupled with other state-of-the-art techniques to systematically characterize the tumor immune microenvironment. Front Mol Biosci 8:673042
Zhang W, Hubbard A, Jones T et al (2017) Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies. Lab Investig 97(7):873–885
Lazarus J, Maj T, Smith JJ et al (2018) Spatial and phenotypic immune profiling of metastatic colon cancer. JCI Insight 3(22):e121932
Carstens JL, Correa de Sampaio P, Yang D et al (2017) Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8:15095
Laberiano-Fernandez C, Hernandez-Ruiz S, Rojas F, Parra ER (2021) Best practices for technical reproducibility assessment of multiplex immunofluorescence. Front Mol Biosci 8:660202
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
McGue, J.J., Edwards, J.J., Griffith, B.D., Frankel, T.L. (2023). Multiplex Fluorescent Immunohistochemistry for Preservation of Tumor Microenvironment Architecture and Spatial Relationship of Cells in Tumor Tissues. In: Kasid, U.N., Clarke, R. (eds) Cancer Systems and Integrative Biology. Methods in Molecular Biology, vol 2660. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3163-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3163-8_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3162-1
Online ISBN: 978-1-0716-3163-8
eBook Packages: Springer Protocols