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Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry

Nature Methods volume 11, pages 417–422 (2014)Cite this article

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Abstract

Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies.

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Figure 1
Figure 2: Validation of imaging mass cytometry.
Figure 3: Representative mass cytometry images of luminal HER2+ breast cancer tissue samples.
Figure 4: Analysis of adherent cells by IFM and imaging mass cytometry.
Figure 5: Imaging mass cytometry analysis of tumor heterogeneity.

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Acknowledgements

We thank M. Storz for preparing the histological slides and the TMA sections; S. Dettwiler, A. Bohnert, A. Fitsche; the entire Trace Element and Micro Analysis group at ETH Zürich for their experimental support and discussions; N. Daga and C. von Mering for their feedback on data analysis; the ETHZ LAC workshop for their support in design and construction of the laser ablation chamber; and the Lehner and Luschnig groups for giving us access to their immunofluorescence microscopes. This work was supported by a Society in Science, The Branco Weiss Fellowship, administered by the ETH Zürich (C.G.); the Swiss National Science Foundation (SNSF) project grants 200021-119779 (H.A.O.W.), 200021-119779 (D. Günther), 31003A-143877 (D. Günther) and 31003A-143877 (B.B.); an ETH Zürich Pioneer Fellowship (H.A.O.W.); the SystemsX PhosphoNet-PPM grant (P.J.W. and B.B.); a Baugarten Foundation grant (SGGP) (P.J.W.); a EU VIGOR++ project FP7/2007-2013, #270379 (P.J.S. and J.M.B.); an SNSF R'Equip grant 316030-139220 (B.B.); an SNSF Assistant Professorship grant PP00P3-144874 (B.B.); a Swiss Cancer League grant (B.B.); and funding from the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement no. 336921 (B.B.).

Author information

Author notes

  1. Charlotte Giesen and Hao A O Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland

    Charlotte Giesen, Denis Schapiro, Nevena Zivanovic, Andrea Jacobs & Bernd Bodenmiller

  2. Department of Chemistry, Swiss Federal Institute of Technology Zürich, Zürich, Switzerland

    Hao A O Wang, Bodo Hattendorf & Detlef Günther

  3. Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland

    Hao A O Wang & Daniel Grolimund

  4. Systems Biology Ph.D. Program, Life Science Zürich Graduate School, ETH Zürich and University of Zürich, Zürich, Switzerland

    Denis Schapiro

  5. Molecular Life Science Ph.D. Program, Life Science Zürich Graduate School, ETH Zürich and University of Zürich, Zürich, Switzerland

    Nevena Zivanovic

  6. Department of Computer Science, Swiss Federal Institute of Technology Zürich, Zürich, Switzerland

    Peter J Schüffler & Joachim M Buhmann

  7. Institute of Surgical Pathology, University Hospital Zürich, Zürich, Switzerland

    Simone Brandt, Zsuzsanna Varga & Peter J Wild

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Contributions

C.G., H.A.O.W., N.Z., A.J., D. Grolimund, Z.V., P.J.W., D. Günther and B.B. designed and performed experiments. H.A.O.W., D. Grolimund and B.H. established the tissue laser ablation system. C.G., H.A.O.W., D.S., N.Z., A.J., P.J.S. and D. Grolimund performed data analysis. S.B., Z.V. and P.J.W. assembled, provided and classified tumor samples. D.S., P.J.S. and J.M.B. arranged image analysis and single-cell segmentation. C.G., H.A.O.W., D.S., N.Z., P.J.W., D. Grolimund, D. Günther and B.B. prepared the figures and wrote the manuscript. D. Günther and B.B. conceived of and supervised the project. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Detlef Günther or Bernd Bodenmiller.

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Competing interests

Fluidigm (formerly DVS Sciences) has an option to license imaging mass cytometry technology from D. Günther represented by ETH Zürich, which includes a related funded research collaboration benefiting the D. Günther and B.B. labs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1–6 and Supplementary Notes 1–3 (PDF 4881 kb)

Supplementary Data

Spade trees generated for the analysed breast cancer images (ZIP 14536 kb)

Supplementary Software

Imaging mass cytometry software (ZIP 100 kb)

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Giesen, C., Wang, H., Schapiro, D. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11, 417–422 (2014). https://doi.org/10.1038/nmeth.2869

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