Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy

Abstract

Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter-observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell-cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost-effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.

Keywords: immunofluorescence; immunohistochemistry; immunotherapy; multiplex; overview.

FIGURE 1

Diagram showing mechanism of each of the mIHC/IF platform. (A) DISCOVERY ULTRA system: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. The HRP is reacted with an appropriate substrate bound to a chromogenic dye, leading to the precipitation of insoluble, coloured precipitates at the site where the antigens are found. (B) Metal‐based IHC techniques such as IMC and MIBI: a primary antibody bound to the target antigen is tagged with a metal isotope of known molecular mass. Analysis is carried out using mass spectrometry in MIBI and laser ablation coupled to mass cytometry in IMC. (C) Vectra: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. A fluorophore‐conjugated tyramide molecule serves as the substrate for HRP, resulting in an antigen‐associated fluorescence signal. (D) Nanostring's DSP: the target antigen will bind the primary antibody which is coupled to a photocleavable oligonucleotide tag. UV light is used to cleave the oligonucleotide tags and is collected using a microcapillary tube and stored in a microplate well. The oligonucleotide tags will bind to the reporter probe via the target‐specific capture probe. Reporter probes are imaged and counted by the nCounter analysis system. Abbreviations: mIHC/IF, multiplex immunohistochemistry/immunofluorescence; HRP, horseradish peroxidase; IHC, immunohistochemistry; IMC, Imaging Mass Cytometry; MIBI, Multiplexed Ion Beam Imaging; DSP, Digital Spatial Profiling

FIGURE 2

Representative mIHC/IF images captured through the Vectra, Chipcytometry, or DISCOVERY ULTRA imaging system. (A) mIHC/IF of pancreatic adenocarcinoma FFPE sections labelled with DAPI (blue), CD73 (green), CD8 (yellow), CD68 (red), FoxP3 (cyan), CD3 (magenta) and CK (orange) were scanned using the Vectra imaging system. (B) Mouse pancreas FFPE sections labelled with CD45 (brown), CD274 (green), CD3e (purple), CD4 (cyan), CD8a (pink), CD11b (yellow), CD31 (dark brown), CD326/EpCAM (red), B220 (orange), F4/80 (blue), NK1.1 (purple), Pan‐CK (maroon), Hoechst 33342 (dark blue) were scanned using the Chipcytometry imaging system. (C) Cholangiocarcinoma FFPE sections labelled with CD20 (blue), CD8 (red), CD68 (turquoise), CD3 (yellow) were scanned using the DISCOVERY ULTRA imaging system. Abbreviations: mIHC/IF, multiplex immunohistochemistry/immunofluorescence; FFPE, Formalin‐Fixed Paraffin‐Embedded; DAPI, 4′,6‐diamidino‐2‐phenylindole

FIGURE 3

Representative IMC images of human tissue sections. Each image depicts the tumor microenvironment with the following immune cell lineages: T cell panel (A; CD45RO depicted in green, CK in cyan, collagen in yellow, CD8 in red, CD4 in magenta, and Ki67 in white), basic lineage panel (B; CD68 in green, CD20 in cyan, PD‐L1 in yellow, VISTA in red, CD3 in magenta, and CD45 in white), and function panel (C; OX40 in green, CD38 in cyan, Ki67 in yellow, ecadherin in red, collagen in magenta, and granzymeB in white). Abbreviations: IMC, imaging mass cytometry; PD‐L1, programmed cell death ligand 1

FIGURE 4

Representative Ultivue's InSituPlex images of human tissue samples labelled with CD8 (green), CD68 (yellow), PD‐L1 (red) and CK/Sox10 (cyan). Whole slide imaging of tonsil section (A), high magnification view of HCC (B), and radioembolization‐treated HCC (C, Y‐90 visible as microspheres). Abbreviations: HCC, hepatocellular carcinoma; PD‐L1, programmed cell death ligand 1