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Multiplexed ion beam imaging of human breast tumors

Nature Medicine volume 20, pages 436–442 (2014)Cite this article

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Abstract

Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

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Figure 1: Workflow summary of MIBI.
Figure 2: Analysis of PBMCs stained with metal-conjugated antibodies using mass cytometry and MIBI.
Figure 3: Ten-color imaging of human breast tumors using MIBI.
Figure 4: Quantitative analysis of tumor immunophenotype.
Figure 5: Composite representation of multidimensional MIBI data using categorical and quantitative colorization.

References

  1. Coons, A.H., Creech, H.J., Jones, R.N. & Berliner, E. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45, 159–170 (1942).

    CAS  Google Scholar 

  2. Rimm, D.L. What brown cannot do for you. Nat. Biotechnol. 24, 914–916 (2006).

    Article  CAS  Google Scholar 

  3. Anagnostou, V.K. et al. Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol. Biomarkers Prev. 19, 982–991 (2010).

    Article  CAS  Google Scholar 

  4. Bordeaux, J. et al. Antibody validation. Biotechniques 48, 197–209 (2010).

    Article  CAS  Google Scholar 

  5. McCabe, A., Dolled-Filhart, M., Camp, R.L. & Rimm, D.L. Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J. Natl. Cancer Inst. 97, 1808–1815 (2005).

    Article  CAS  Google Scholar 

  6. Hasui, K. et al. Double autoimmunostaining with glycine treatment. J. Histochem. Cytochem. 51, 1169–1176 (2003).

    Article  CAS  Google Scholar 

  7. Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

    Article  CAS  Google Scholar 

  8. Lou, X. et al. Polymer-based elemental tags for sensitive bioassays. Angew. Chem. Int. Edn Engl. 46, 6111–6114 (2007).

    Article  CAS  Google Scholar 

  9. Ornatsky, O.I. et al. Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom. 23, 463–469 (2008).

    Article  CAS  Google Scholar 

  10. Ornatsky, O. et al. Highly multiparametric analysis by mass cytometry. J. Immunol. Methods 361, 1–20 (2010).

    Article  CAS  Google Scholar 

  11. Bandura, D.R. et al. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal. Chem. 81, 6813–6822 (2009).

    Article  CAS  Google Scholar 

  12. Blow, N. Tissue preparation: tissue issues. Nature 448, 959–963 (2007).

    Article  Google Scholar 

  13. Lechene, C. et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J. Biol. 5, 20 (2006).

    Article  Google Scholar 

  14. Senyo, S.E. et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature 493, 433–436 (2013).

    Article  CAS  Google Scholar 

  15. Steinhauser, M.L. et al. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism. Nature 481, 516–519 (2012).

    Article  CAS  Google Scholar 

  16. Williams, P. Biological imaging using secondary ions. J. Biol. 5, 18 (2006).

    Article  Google Scholar 

  17. Gordon, A. et al. Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat. Methods 4, 175–181 (2007).

    Article  CAS  Google Scholar 

  18. Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  Google Scholar 

  19. Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  Google Scholar 

  20. Bodo, J., Durkin, L. & Hsi, E.D. Quantitative in situ detection of phosphoproteins in fixed tissues using quantum dot technology. J. Histochem. Cytochem. 57, 701–708 (2009).

    Article  CAS  Google Scholar 

  21. Camp, R.L., Chung, G.G. & Rimm, D.L. Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat. Med. 8, 1323–1327 (2002).

    Article  CAS  Google Scholar 

  22. Tsurui, H. et al. Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. J. Histochem. Cytochem. 48, 653–662 (2000).

    Article  CAS  Google Scholar 

  23. Glass, G., Papin, J.A. & Mandell, J.W. SIMPLE: a sequential immunoperoxidase labeling and erasing method. J. Histochem. Cytochem. 57, 899–905 (2009).

    Article  CAS  Google Scholar 

  24. Wählby, C., Erlandsson, F., Bengtsson, E. & Zetterberg, A. Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry 47, 32–41 (2002).

    Article  Google Scholar 

  25. Pirici, D. et al. Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype. J. Histochem. Cytochem. 57, 567–575 (2009).

    Article  CAS  Google Scholar 

  26. Friedenberger, M., Bode, M., Krusche, A. & Schubert, W. Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures. Nat. Protoc. 2, 2285–2294 (2007).

    Article  CAS  Google Scholar 

  27. Schubert, W. et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24, 1270–1278 (2006).

    Article  CAS  Google Scholar 

  28. Schubert, W., Gieseler, A., Krusche, A. & Hillert, R. Toponome mapping in prostate cancer: detection of 2000 cell surface protein clusters in a single tissue section and cell type specific annotation by using a three symbol code. J. Proteome Res. 8, 2696–2707 (2009).

    Article  CAS  Google Scholar 

  29. Wu, B. & Becker, J.S. Imaging of elements and molecules in biological tissues and cells in the low-micrometer and nanometer range. Int. J. Mass Spectrom. 307, 112–122 (2011).

    Article  CAS  Google Scholar 

  30. Seeley, E.H. & Caprioli, R.M. Imaging mass spectrometry: Towards clinical diagnostics. Proteomics Clin. Appl. 2, 1435–1443 (2008).

    Article  CAS  Google Scholar 

  31. Aerni, H.-R., Cornett, D.S. & Caprioli, R.M. High-throughput profiling of formalin-fixed paraffin-embedded tissue using parallel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Anal. Chem. 81, 7490–7495 (2009).

    Article  CAS  Google Scholar 

  32. Caprioli, R.M. Perspectives on imaging mass spectrometry in biology and medicine. Proteomics 8, 3679–3680 (2008).

    Article  CAS  Google Scholar 

  33. Giesen, C. et al. Multiplexed immunohistochemical detection of tumor markers in breast cancer tissue using laser ablation inductively coupled plasma mass spectrometry. Anal. Chem. 83, 8177–8183 (2011).

    Article  CAS  Google Scholar 

  34. Smith, N.S., Tesch, P.P., Martin, N.P. & Kinion, D.E. A high brightness source for nano-probe secondary ion mass spectrometry. Appl. Surf. Sci. 255, 1606–1609 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank N. Hubbard, C. Espiritu, S. Rost, L. Rangell and the Genentech Human Tissue Lab for assistance in preparing and processing tissue sections. We also thank A. Jager for technical support with CyTOF and antibody labeling. M.A. is supported by the Stanford Molecular Imaging Scholars Program through the US National Institutes of Health (NIH, 5R25CA11868107). S.C.B. is supported by the Damon Runyon Cancer Research Foundation Fellowship (DRG-2017-09) and NIH (1K99 GM104148-01). R.F. is supported by Northrop-Grumman Corporation (7500108142 BISC). This work was supported by a US National Science Foundation equipment grant (0922648) to the Stanford Nano Center for the NanoSIMS 50L analytical system used in the work here. This work was also supported by grants (to the Nolan lab) from the NIH (0158 G KB065, 1R01CA130826, 5U54CA143907, HHSN272200700038C, N01-HV-00242, 41000411217, 5-24927, P01 CA034233-22A1, P01 CA034233-22A1, PN2EY018228, RFA CA 09-009, RFA CA 09-011, U19 AI057229 and U54CA149145), the California Institute for Regenerative Medicine (DR1-01477 and RB2-01592), the European Commission (HEALTH.2010.1.2-1), the US FDA (HHSF223201210194C: BAA-12-00118), the US Department of Defense (W81XWH-12-1-0591 OCRP-TIA NWC) and the Entertainment Industry Foundation.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Baxter Laboratory in Stem Cell Biology, Stanford University, Stanford, California, USA

    Michael Angelo, Sean C Bendall, Rachel Finck, Matthew B Hale & Garry P Nolan

  2. Department of Laboratory Medicine, University of California–San Francisco, San Francisco, California, USA

    Michael Angelo

  3. Department of Materials Science and Engineering, Stanford University, Stanford, California, USA

    Chuck Hitzman

  4. Department of Pathology and Laboratory Medicine, University of California–Davis, Health System, Sacramento, California, USA

    Alexander D Borowsky & Richard M Levenson

  5. Department of Pathology, Genentech, South San Francisco, California, USA

    John B Lowe & Scot D Liu

  6. Department of Pathology, Stanford University, Stanford, California, USA

    Shuchun Zhao & Yasodha Natkunam

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  1. Michael Angelo
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Contributions

M.A., S.C.B. and R.F. conducted experiments and wrote the manuscript. M.B.H. designed and fabricated reagents. C.H. assisted in data acquisition and experimental design. A.D.B. and R.M.L. prepared tissue sections, performed IHC and assisted in writing the manuscript. J.B.L., S.D.L., S.Z. and Y.N. prepared tissue sections, performed IHC and assisted in optimizing protocols used in MIBI analysis. G.P.N. assisted in experimental design and wrote the manuscript.

Corresponding author

Correspondence to Garry P Nolan.

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Competing interests

G.P.N. has personal financial interest in the company DVS Sciences, the manufacturer of the mass cytometer and antibody conjugation reagents used in this manuscript.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2. (PDF 2647 kb)

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Angelo, M., Bendall, S., Finck, R. et al. Multiplexed ion beam imaging of human breast tumors. Nat Med 20, 436–442 (2014). https://doi.org/10.1038/nm.3488

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