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Multiplexed ion beam imaging of human breast tumors


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.


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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.

Author information

Authors and Affiliations



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).

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