Next-Generation Pathology Using Multiplexed Immunohistochemistry: Mapping Tissue Architecture at Single-Cell Level

Abstract

Single-cell omics aim at charting the different types and properties of all cells in the human body in health and disease. Over the past years, myriads of cellular phenotypes have been defined by methods that mostly required cells to be dissociated and removed from their original microenvironment, thus destroying valuable information about their location and interactions. Growing insights, however, are showing that such information is crucial to understand complex disease states. For decades, pathologists have interpreted cells in the context of their tissue using low-plex antibody- and morphology-based methods. Novel technologies for multiplexed immunohistochemistry are now rendering it possible to perform extended single-cell expression profiling using dozens of protein markers in the spatial context of a single tissue section. The combination of these novel technologies with extended data analysis tools allows us now to study cell-cell interactions, define cellular sociology, and describe detailed aberrations in tissue architecture, as such gaining much deeper insights in disease states. In this review, we provide a comprehensive overview of the available technologies for multiplexed immunohistochemistry, their advantages and challenges. We also provide the principles on how to interpret high-dimensional data in a spatial context. Similar to the fact that no one can just "read" a genome, pathological assessments are in dire need of extended digital data repositories to bring diagnostics and tissue interpretation to the next level.

Keywords: methods for spatial profiling; multiplexed immunofluorescencence and immunohistochemistry; single-cell ‘omics; spatial profiling; tissue architecture analysis.

Figure 1

Schematic overview of the currently available methods for multiplexed immunohistochemistry (IHC). (A) Currently, the most common approach for multiplexed IHC makes use of fluorescently labelled probes, which are either directly coupled to the primary antibody or indirectly provided by a secondary antibody, that are detected in a cyclic fashion consisting of a staining protocol, followed by tissue imaging and signal removal. (B) In contrast to cyclic methods, single-step spectral methods detect all dyes in the tissue simultaneously: these can either be provided by directly labelled antibodies that are all simultaneously present in the tissue section or by the cyclic generation of TSA precipitates which are subsequently spectrally unmixed in a single imaging step. (C) Antibodies can also be detected by covalently linked nucleotide labels to which fluorescently labelled probes are hybridized in a cyclic fashion for which each cycle gets imaged. (D) Non-fluorescent mIHC methods involve the cyclic generation of chromogenic substrates that are washed away following an imaging step in between each cycle. (E) For imaging mass cytometry (IMC), antibodies are labelled with metal isotopes which are detected by the local vaporization of the metal ions by a UV laser, following which the present isotopes are resolved using atomic spectrometry. (F) Finally, nucleotide labelled antibodies can be detected by removing the nucleotide labels from the antibodies using a laser beam, following which the nucleotides that were collected from a precise region of interest are sequenced to quantify the amount of available proteins in that region.

Figure 2

Schematic overview of the required steps for downstream image analysis using the most commonly used fluorescent, cyclic methods for multiplexed IHC. Images are collected across multiple cycles but still need to be cleaned (QC), corrected (PP), registered/aligned (REG), autofluorescence removed (AF), segmented (SEG), feature extracted (FE), phenotypically annotated (PI), and spatially resolved (SA).

Figure 3

The bibliometric map of multiplexed IHC over the years. Using VOS Viewer, a software tool for constructing and visualizing the bibliometric network related to “multiplexed immunohistochemistry” on PUBMED (see Methods), we observed a shift from 2010 where technology development started (blue circles), to its use to unravel complex cellular networks in 2020 (yellow areas) with a strong focus on immuno-oncology and T-cell biology.