Multiplexing Immunohistochemistry
Multiplexing immunohistochemistry offers an unmatched insight into spatial proteomics in a variety of cell populations and bridges the gap between high throughput and single cell technologies.
What is Immunohistochemistry?
Immunohistochemistry (IHC) represents a powerful tool used for the identification of target proteins in the context of the cell or tissue analyzed. This type of in situ analysis is effective in assessing the localization of proteins in the cell, comparing the expression of a protein in a heterogeneous cell population, and can be used to identify sub-populations of cells based on the expression of specific markers.
The Limitations of IHC
The inability to label more than one marker per tissue section is the most important limitation of IHC. Another drawback of IHC‐based biomarker assessment is high inter‐observer variability. Thus, while IHC remains a highly practical and cost‐effective diagnostic and prognostic method, this single‐marker method cannot tell the whole story of complex immune microenvironment.
What is Multiplexing Immunohistochemistry?
Multiplexing Immunohistochemistry (mIHC) technologies, which allow the simultaneous detection of multiple markers on a single tissue section, have been introduced and adopted in both research and clinical settings in response to increased demand for improved techniques.
A number of highly multiplexed tissue imaging technologies have also emerged, permitting comprehensive studies of cell composition, functional state and cell‐cell interactions which suggest improved diagnostic benefit. These novel imaging techniques are based on cyclic immunofluorescence, tyramide‐based mIHC, epitope‐targeted mass spectrometry, or RNA detection. Such techniques provide a comprehensive view of marker distribution and tissue composition, and are poised to solve major questions surrounding the pathogenesis of various complex disorders.
In terms of the more advanced, higher-level applications of mIHC, there are various approaches relying on distinct principles. These can be classified into five classes: stain removal, fluorophore inactivation, multiplexed signal amplification, DNA barcoding, and mass cytometry.
Stain Removal Technologies
One of the most commonly used methods of mIHC is stain removal. Stain removal technologies (sometimes called dye cycling) refer to a class of protocols that rely on a cycle of antibody staining, image capture, removal of stain, and re-staining to greatly increase the number of markers that can be tested in a single experiment.
Multiepitope-ligand cartography (MELC)
MELC is an automated method to measure up to thousands of different classes, parts, or groups of molecules in liquid or solid samples, especially in cells and tissue specimens.
MELC has been frequently used to study the intricacies of the immune system. However, photobleaching can only be applied to the field of view, limiting the depth of information gathered in one round of cycling.
Sequential immuno-peroxidase labelling and erasing (SIMPLE)
SIMPLE is also based on stain removal technology, using the alcohol-soluble red peroxidase substrate 3-amino-9-ethylcarbazole (AEC) for at least parallel five markers visualization.
SIMPLE has been applied for probing the immune system, often in the context of cancer. Additionally, it helped to indicate the therapeutic response to vaccination therapy in pancreatic ductal adenocarcinoma.
Iterative bleaching extends multiplexity (IBEX)
IBEX is a recently emerging technology, which involves iterative immunolabeling and chemical bleaching method, enabling multiplexed imaging in various tissues.
IBEX enables high-resolution imaging of over 65 parameters simultaneously without physical damage. Therefore, it is a suitable method for revealing the complex cellular architecture and tumor immune interactions under a spatial context, contributing to tissue physiology and pathology.
Fluorophore Inactivation Technologies
The principle of this technology is roughly similar to stain removal technologies. However, it does not rely on using different pH conditions, denaturation, or photobleaching to remove a stain, but on chemical inactivation to eliminate the fluorophore.
Multiplexed fluorescence microscopy method (MxIF)
MxIF is an imaging platform that enables 60 directly labeled antibodies to be applied to a single tissue section. This method can provide quantitative, single-cell, and subcellular characterization of multiplexed molecular targets in FFPE tissue.
Cyclic immunofluorescence (CycIF)
CycIF is a public-domain technology, which as the name suggests, involves repeated cycles of immunofluorescence staining and fluorophore inactivation. Compared to MxIF, it allows to use general reagents and commercial antibodies without cyanine modification.
For investigating responsiveness and resistance to therapy, a tissue-based cyclic immunofluorescence (t-CyCIF) method has been described in 2018. t-CyCIF allowed detection of over 60 different proteins in normal and tumor tissue samples, giving an efficient method for pre-clinical and clinical research.
Multiplexed Signal Amplification
One of the problems with detecting multiple markers in a sample is that sometimes the epitopes are present at a low copy number. Multiplexed signal amplification methods aim to address this need, providing strong signals for imaging even small amounts of protein.
Here, tyramide signal amplification (TSA) is a commonly used one.
Tyramide signal amplification (TSA)
TSA is an enzyme-mediated method that catalyzes the deposition of tyramide from low to large amount in an immunoassay system. Tyramide can be biotinylated or labeled with fluorescent dye and catalyzed by streptavidin-horseradish peroxidase enzyme (HRP). Subsequently, multiple tyramide-labeled molecules are laid down at the site of the epitope. TSA can provide a systematic evaluation of different processes in different tumor tissues.
DNA Barcoding Technologies
Another approach to multiplexing is using the innate properties and specificities of DNA itself. These technologies take advantage of DNA's sequence-specific binding properties to generate highly specific staining which is intrinsically applicable to multiplexing and mapping.
DNA exchange imaging (DEI)
DEI builds upon a previously reported super-resolution technology that permitted multiplexed imaging of DNA in situ or in vitro and adapts the technique to more complex cell populations and tissues. One of the major advantages of DEI is its speed, overcoming the need for multiple cycles of imaging over several hours.
Co-detection by Indexing (CODEX)
CODEX is another DNA barcoding technique for highly multiplexed cytometric imaging. Unlike the other methods, the antibodies are labeled with DNA oligonucleotides, instead of fluorophores or rare metal elements.
Signal amplification by exchange reaction (SABER)
One limitation of DEI and CODEX is that DNA strands, which are conjugated to primary antibodies directly, lack secondary antibodies for signal amplification, especially in low abundance target detection for tissues. To overcome this limitation, SABER method was developed.
Based on SABER technology, immunostaining with signal amplification by exchange reaction (Immuno-SABER) can achieve highly multiplexed signal amplification in situ without enzymatic reactions.
Mass Cytometry
Mass cytometry is another example of a non-microscopy-based method of quantifying multiple biomarkers, that allows users to carry out multiplexing.
Based on mass cytometry technology, several methods have been developed such as imaging mass cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI).
Imaging mass cytometry (IMC)
IMC is an optimized mass cytometry, which is applied in immunocytochemistry and immunohistochemistry. Owning to a high-resolution laser ablation system, IMC can reveal the spatial information, allow the description of cell subtypes and cell-cell interactions and emphasize tumor heterogeneity.
Multiplexed ion beam imaging (MIBI)
MIBI is very similar to IMC but utilizes secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters.