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Multiplexed immunohistochemical (IHC) analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples allows researchers to study the spatial relationships between different cell phenotypes in situ. Nowhere is this more powerful than within the tumor microenvironment, where such information could provide unique insight to guide prognosis and therapy. This is highlighted by a growing number of publications describing the use of multiplex IHC within oncology research.
Multiplex capabilities can increase the rate of discovery of new biomarkers and therapeutically targetable pathways.
By enabling deeper analysis of the tumor microenvironment than permitted by single-plex techniques, multiplex imaging has broad ranging benefits for researchers, clinicians and drug developers. From a clinical perspective, simultaneous analysis of multiple biomarkers can be more reliable for stratifying patients and predicting patient response than using a single predictive biomarker. For basic research, multiplex capabilities can increase the rate of discovery of new biomarkers and therapeutically targetable pathways.
Driven by these needs, various antibody-based approaches have been developed to support multiplex tissue imaging. Ranging from sequential staining protocols through to advanced mass spectrometry techniques, methods have evolved that offer improved sensitivity and an increased degree of multiplexing. While each approach has its own advantages and considerations for success, a common factor is that the quality of the data obtained relies on the antibodies that are used.
Sequential colorimetric and immunofluorescent staining
In its simplest format, IHC staining uses a primary antibody to detect the target of interest, and an anti-species secondary antibody labeled with an enzyme or fluorescent dye to generate a visible signal. An enzyme-based chromogenic IHC protocol usually provides a single readout, whereas fluorometric IHC (also known as IHC-IF and multispectral IF) typically combines two or three antibodies with a nuclear stain. Experimental timelines can be shortened and simplified by incorporating directly conjugated primary antibodies into staining protocols.
By carrying out sequential staining cycles based on these methods, it is possible to achieve multiplex tissue imaging. Various approaches have been developed for sequential immunofluorescence, many of which involve the addition of pairs of fluorophore-labeled antibodies to the sample material in consecutive rounds of staining. Included among these, MultiOmyx™ hyperplexed immunofluorescence uses a dye inactivation chemistry between each iteration to study up to 60 biomarkers on a single slide, while iterative indirect immunofluorescence imaging (4i) employs chemical antibody elution to generate 40-plex readouts.
Experimental timelines can be shortened and simplified by incorporating directly conjugated primary antibodies into staining protocols.
Frequently used to enhance the detection of low abundance targets, Opal™ technology relies on HRP-labeled secondary antibodies to catalyze the reaction of tyramide signal amplification. Once the fluorescently labeled tyramide has covalently bound to tyrosine residues on and around the protein of interest, heat retrieval is used to remove the antibodies without disturbing the Opal™ fluorescent signal; the cycle can then be repeated. The Opal™ workflow has been optimized for use on the Leica Biosystems BOND RX and can be integrated with Phenoptics™ instrumentation to increase throughput.
Also relying on iterative addition of unlabeled antibodies, Roche Diagnostics’ DISCOVERY ULTRA is another automated IHC platform that uses heat inactivation to detach antibodies between sequential rounds of staining. This system is widely used for multiplex IHC with DISCOVERY chromogens, a product range that includes specialized translucent reagents for colocalization of antigenic targets; this is achieved through the formation of an additional color where there is overlap with another chromogen.
New methods for labeling primary antibodies
The development of various enabling technologies has seen increased use of antibody-oligonucleotide conjugates for IHC, an approach that can greatly increase assay sensitivity. Ultivue’s InSituPlex® technology and Akoya Biosciences’ CODEX® technology both rely on oligo-labeled antibodies for tissue imaging. During the Ultivue method, antibodies against four different targets are added to the sample simultaneously and the conjugated oligos are subsequently amplified; target detection requires the addition of fluorescently labeled complementary DNA probes. The CODEX® approach has similarities in that it also uses dye-labeled reporters; however, it will function to a higher plex through multiple rounds of staining and can be integrated with Opal™ to streamline workflows.
Use of antibody-oligonucleotide conjugates for IHC can greatly increase assay sensitivity.
An alternative to using antibody-oligonucleotide conjugates is to incorporate hapten-labeled antibodies into multiplex tissue imaging protocols. Like antibody-oligonucleotide conjugates, these reagents eliminate the need to use primary antibodies of different species or isotypes to avoid cross-reactivity with labeled secondary antibodies. Cell IDx’ UltraPlex multiplexing technology employs panels of antibody-hapten conjugates to stain tissue samples, using anti-hapten secondaries to detect antibody binding. Through tissue alignment of serial sections, researchers can analyze more than four different biomarkers plus a nuclear stain with this method.
Highly multiplexed staining methods
Although many of the sequential staining methods described here also deliver simultaneous antigen detection to some extent, other approaches are available to multiplex a far greater number of targets at the same time. These provide a highly detailed ‘snapshot’ of the tissue sample under investigation; however, many simultaneous staining methods require highly specialized instrumentation.
NanoString’s digital spatial profiling (DSP) workflow uses an nCounter® to achieve detection of up to 96 proteins at the same time with antibodies that are coupled to photocleavable oligonucleotide tags. After antibody binding, UV light is used to release the tags, which are quantified before being mapped back to the tissue location. This method provides a comprehensive profile of analyte abundance.
Many simultaneous staining methods require highly specialized instrumentation.
Imaging mass cytometry™ (IMC™) from Fluidigm and multiplexed ion beam imaging (MIBI™) technology from IONpath instead use a mass spectrometer to obtain multiple labels within an area of the tissue. Both techniques rely on antibodies tagged with metal isotopes of known molecular mass. By greatly reducing the spillover issue inherent to fluorescent imaging, these methods increase the degree of multiplexing by allowing more antibodies to be combined in a single experiment, routinely achieving simultaneous detection of up to 40 markers.
High-quality, application-appropriate antibodies
Whatever approach to multiplex IHC tissue imaging is chosen, high-quality, reliable antibodies are essential. Due to the extensive and rigorous validation that many researchers employ when selecting antibody clones for their multiplex IHC panels, batch-to-batch consistency is crucial. Not only should a candidate antibody be recommended for IHC, and tested in relevant samples but, if they are to be conjugated, should be supplied in a carrier-free format since common additives such as BSA and glycerol are detrimental to the conjugation reaction.
Abcam have almost 4,000 IHC-validated RabMAb® rabbit monoclonal antibodies, many in recombinant format for unparalleled batch-to-batch consistency. All are available in carrier-free format. Find out more.