Multiplexed ion beam imaging of human breast tumors

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.

Figure 1. Workflow summary of multiplexed ion beam imaging (MIBI)

Biological specimens, such as FFPE tissue or cell suspensions, are immobilized on a conductive substrate, such as indium tin oxide coated glass or silicon wafer. Samples are subsequently stained with antibodies conjugated to unique transition element isotope reporters, dried, and loaded under vacuum for MIBI analysis. The sample surface is rasterized with a primary ion beam (O^-) that sputters the antibody-specific isotope reporters native to the sample surface as secondary ions. Metal conjugated antibodies are quantified via replicate scans of the same field of view, where up to seven metals reporters are measured with each scan, and the detectors are mass calibrated in between each scan cycle. Regions of interest demarcating nuclear and cytosolic compartments of each cell are integrated, tabulated, and categorized. Composite images comprised of pseudo-colored categorical features and quantitative three-color overlays are constructed to summarize multidimensional expression data.

Figure 2. Analysis of PBMCs stained with metal-conjugated antibodies using mass cytometry and MIBI

(a) PBMCs stained with seven antibodies were immobilized on a silicon wafer and imaged using MIBI. Single cell regions of interest were segmented using CD45 surface expression and integrated for each antibody. (b, c) Hierarchical gating of the resultant data yielded comparable values for seven cell populations relative to those found by mass cytometry. Biaxial plots are arcsinh(x/5) scaled. (d) Pearson correlation of the relative abundance of each cell population demonstrated strong agreement between the two methods (r = 0.98, P < 0.0001, two-tailed t-test).

Figure 3. 10-color imaging of human breast tumors using MIBI

(a) Avidity of primary antibodies is unaffected by metal-conjugation. To access the effect of metal conjugation on antibody avidity, immunoperoxidase staining of serial sections from a single human breast tumor were stained with metal-conjugated or unmodified primary antibodies for Ki67 or ER-alpha. Positive-staining nuclei of comparable intensity were present in similar numbers when using metal-conjugated or unmodified primary antibodies. (b) Visual representation of MIBI data. Single channel ion data can be color mapped and merged to construct pseudo-brightfield or pseudo-darkfield images resembling conventional immunoperoxidase or immunofluorescence staining, respectively. (c) 10-color imaging of human breast tumors. FFPE tissue sections from three different patients were analyzed using MIBI. HER2, ER, and PR are expressed appropriately with respect to the known immunophenotype of each specimen. ER, PR, and Ki67 demonstrate well-demarcated nuclear positivity, while e-cadherin, actin, HER2, and keratin expression is appropriately membranous. Field of view = 80 μm.

Figure 4. Quantitative analysis of tumor immunophenotype

(a) For quantitative single cell analysis, ion images are segmented into ROIs demarcating nuclear and cytoplasmic compartments. (b) Examination of the resultant data using conventional biaxial scatter plots demonstrates quantitative expression patterns matching the known immunophenotype of each respective tumor. Each point represents the mean pixel intensity for each respective cell ROI. Biaxial plots are log scaled. (c) Comparison of ER staining by IHC and MIBI. ER staining intensity of a cohort of breast tumors analyzed using IHC were compared with corresponding values attained using MIBI. Linear regression analysis comparing mean intensities (Top) and H-scores (Bottom) using the two methods demonstrates strong, robust agreement between the two methods (r = 0.99, P < 0.0001).

Figure 5. Composite representation of multidimensional MIBI data using categorical and quantitative colorization

(a) Quantitative colorization of cytoplasmic features. Green-encoded e-cadherin, red-encoded actin, and blue-encoded vimentin channels were merged to generate a quantitative representation of protein expression and colocalization. (b) Categorical colorization of nuclei. Subpopulations of ER⁺PR⁺Ki67⁺ positive or ER⁺PR⁺ positive nuclei are pseudo-colored yellow or aqua, respectively. (c) Multidimensional data are summarized in a composite image illustrating quantitative and categorical expression patterns.