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Multiplex protein imaging in tumour biology

Nature Reviews Cancer volume 24, pages 171–191 (2024)Cite this article

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Abstract

Tissue imaging has become much more colourful in the past decade. Advances in both experimental and analytical methods now make it possible to image protein markers in tissue samples in high multiplex. The ability to routinely image 40–50 markers simultaneously, at single-cell or subcellular resolution, has opened up new vistas in the study of tumour biology. Cellular phenotypes, interaction, communication and spatial organization have become amenable to molecular-level analysis, and application to patient cohorts has identified clinically relevant cellular and tissue features in several cancer types. Here, we review the use of multiplex protein imaging methods to study tumour biology, discuss ongoing attempts to combine these approaches with other forms of spatial omics, and highlight challenges in the field.

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Fig. 1: Multiplex protein imaging can interrogate a tumour and its microenvironment across scales.
Fig. 2: Multiplex protein imaging can be applied to clinical cohorts.
Fig. 3: An overview of spatial analysis approaches for multiplex protein imaging data.
Fig. 4: The principles of single-cell multiplex protein imaging methods.
Fig. 5: Canonical pipeline for multiplex protein image processing.
Fig. 6: The principles of spatial multi-omics techniques.

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Acknowledgements

We thank Nils Eling and Daniel Schulz for critical reading and feedback on the manuscript. We thank all Bodenmiller laboratory members for helpful discussions. B.B. was funded by two SNSF grants (310030_205007, 316030_213512), an NIH grant (UC4 DK108132), the CRUK IMAXT Grand Challenge, and the European Research Council (ERC) under the European Union’s Horizon 2020 Program under the ERC grant agreement no. 866074 (“Precision Motifs”).

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Authors and Affiliations

  1. University of Zurich, Department of Quantitative Biomedicine, Zurich, Switzerland

    Natalie de Souza, Shan Zhao & Bernd Bodenmiller

  2. ETH Zurich, Institute of Molecular Systems Biology, Zurich, Switzerland

    Natalie de Souza

  3. ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland

    Natalie de Souza, Shan Zhao & Bernd Bodenmiller

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  1. Natalie de Souza
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Contributions

N.d.S. and S.Z. researched data for the article. All authors contributed substantially to discussion of the content. N.d.S. wrote and revised the article. S.Z. prepared and revised the figures and tables with input from the other authors. N.d.S. and B.B. reviewed and edited the manuscript before submission.

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Correspondence to Bernd Bodenmiller.

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Competing interests

B.B. has founded and is a shareholder and member of the board of Navignostics, a precision oncology spin-off from the University of Zurich. N.d.S. and S.Z. declare no competing interests.

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Nature Reviews Cancer thanks Sizun Jiang who co-reviewed with Hendrik Michel, Christian Schürch and Sean Bendall for their contribution to the peer review of this work.

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Related links

Human protein atlas: https://www.proteinatlas.org/

Napari: https://zenodo.org/record/7276432

Online documentation of IMC data analysis: https://bodenmillergroup.github.io/IMCDataAnalysis/

Supplementary information

Glossary

Cell segmentation

An image processing step that delineates the boundaries of individual cells.

Chromogen

A compound that can be converted into a coloured product that can be detected by light microscopy.

Co-detection by indexing

(CODEX). A highly multiplex protein imaging technique that uses iterative hybridization and stripping of fluorophore-tagged DNA oligonucleotide probes to image samples stained with DNA barcode-tagged antibodies.

Cyclic immunofluorescence

(CycIF). A highly multiplex imaging technique using iterative staining with fluorophore-tagged antibodies coupled with chemical inactivation of fluorophores between staining cycles to build a multiplex image of a labelled sample.

Digital spatial profiling

(DSP). A highly multiplex profiling technique for mRNA or protein that uses patterned light to release UV-photocleavable oligonucleotide tags attached to antibodies or to RNA probes in a defined spatial region, followed by sequencing or single molecule counting as a readout.

Dimensionality reduction

A data-processing approach whereby high-dimensional data are projected into a low number of dimensions represented by a smaller subset of variables with essentially the same information content as the full measured set.

Expansion microscopy

An approach that uses polymer-based physical expansion of a sample to improve the resolution of fluorescence microscopy beyond the diffraction limit of light.

Haptens

Small molecules that are not intrinsically antigenic but become so in combination with a macromolecule such as a protein.

Image registration

Data processing steps that bring two or more different images into a single coordinate system such that the images can be aligned.

Imaging mass cytometry

(IMC). A highly multiplex protein or RNA imaging technique that couples mass cytometry by time of flight with high-resolution laser ablation to image samples labelled with metal isotope reporter-tagged antibodies.

Imputation

An approach for handling missing data, typically by replacement with substitute values.

Multiplexed ion beam imaging

(MIBI). A highly multiplex protein imaging technique that uses secondary ion mass spectrometry to image samples labelled with metal isotope reporter-tagged antibodies.

Raman microscopy

Spatially resolved chemical analysis of a sample based on the detection of vibrational modes by scattered light.

Signal amplification by exchange reaction

(SABER). A signal amplification approach based on hybridization of imager DNA strands to concatemerized DNA barcodes assembled on antibodies used to label a sample; compatible with fluorescence and mass cytometric multiplex imaging.

Spillover correction

Data processing steps that compensate for fluorescent or metal signals from one channel that are detected artefactually in a different channel.

Synchrotron

A machine that accelerates charged particles (electrons) to almost the speed of light and thereby generates very intense light, mostly in the X-ray region.

Tertiary lymphoid structures

(TLS). Structured multicellular aggregates of immune cells found outside of lymph nodes, in peripheral tissue, and that reflect inflammatory signalling in the tissue.

Tyramide-based amplification

A signal amplification approach in which horseradish peroxidase (typically coupled to an antibody) catalyses the conversion of labelled tyramide to a reactive molecule that covalently labels nearby proteins at high density.

Voxel gating

Data processing steps to identify a cell of interest based on selecting voxels that are positive for expected markers and negative for incorrect or irrelevant markers.

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de Souza, N., Zhao, S. & Bodenmiller, B. Multiplex protein imaging in tumour biology. Nat Rev Cancer 24, 171–191 (2024). https://doi.org/10.1038/s41568-023-00657-4

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