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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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).
Rimm, D.L. What brown cannot do for you. Nat. Biotechnol. 24, 914–916 (2006).
Anagnostou, V.K. et al. Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol. Biomarkers Prev. 19, 982–991 (2010).
Bordeaux, J. et al. Antibody validation. Biotechniques 48, 197–209 (2010).
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).
Hasui, K. et al. Double autoimmunostaining with glycine treatment. J. Histochem. Cytochem. 51, 1169–1176 (2003).
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).
Lou, X. et al. Polymer-based elemental tags for sensitive bioassays. Angew. Chem. Int. Edn Engl. 46, 6111–6114 (2007).
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).
Ornatsky, O. et al. Highly multiparametric analysis by mass cytometry. J. Immunol. Methods 361, 1–20 (2010).
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).
Blow, N. Tissue preparation: tissue issues. Nature 448, 959–963 (2007).
Lechene, C. et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J. Biol. 5, 20 (2006).
Senyo, S.E. et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature 493, 433–436 (2013).
Steinhauser, M.L. et al. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism. Nature 481, 516–519 (2012).
Williams, P. Biological imaging using secondary ions. J. Biol. 5, 18 (2006).
Gordon, A. et al. Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat. Methods 4, 175–181 (2007).
Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).
Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).
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).
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).
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).
Glass, G., Papin, J.A. & Mandell, J.W. SIMPLE: a sequential immunoperoxidase labeling and erasing method. J. Histochem. Cytochem. 57, 899–905 (2009).
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).
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).
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).
Schubert, W. et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24, 1270–1278 (2006).
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).
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).
Seeley, E.H. & Caprioli, R.M. Imaging mass spectrometry: Towards clinical diagnostics. Proteomics Clin. Appl. 2, 1435–1443 (2008).
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).
Caprioli, R.M. Perspectives on imaging mass spectrometry in biology and medicine. Proteomics 8, 3679–3680 (2008).
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).
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).
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.
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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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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