Immunohistochemistry (IHC) is a tool for visualizing protein expression employed as part of the diagnostic work-up 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 (FFPE) human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI will provide new insights by integrating tissue microarchitecture with highly multiplexed protein expression patterns, and will be valuable for basic research, drug discovery and clinical diagnostics.
In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman and Powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples, and a five-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely-packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4–5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl−:CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing, the massive bioaccumulation of a weakly basic drug.
Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the “tag” and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.
Part of developing therapeutics is the need to identify patients who will respond to treatment. For HER2-targeted therapies, such as trastuzumab, the expression level of HER2 is used to identify patients likely to receive benefit from therapy. Currently, chromogenic immunohistochemistry on patient tumor tissue is one of the methodologies used to assess the expression level of HER2 to determine eligibility for trastuzumab. However, chromogenic staining is fraught with serious drawbacks that influence scoring, which is additionally flawed due to the subjective nature of human/pathologist bias. Thus, to advance drug development and precision medicine, there is a need to develop technologies that are more objective and quantitative through the collection and integration of larger data sets. In proof of concept experiments, we show multiplexed ion beam imaging (MIBI), a novel imaging technology, can quantitate HER2 expression on breast carcinoma tissue with known HER2 status and those values correlate with pathologist-determined IHC scores. The same type of quantitative analysis using the mean pixel value of five individual cells and total pixel count of the entire image was extended to a blinded study of breast carcinoma samples of unknown HER2 scores. Here, a strong correlation between quantitation of HER2 by MIBI analysis and pathologist-derived HER2 IHC score was identified. In addition, a comparison between MIBI analysis and immunofluorescence-based automated quantitative analysis (AQUA) technology, an industry-accepted quantitation system, showed strong correlation in the same blind study. Further comparison of the two systems determined MIBI was comparable to AQUA analysis when evaluated against pathologist-determined scores. Using HER2 as a model, these data show MIBI analysis can quantitate protein expression with greater sensitivity and objectivity compared to standard pathologist interpretation, demonstrating its potential as a technology capable of advancing cancer and patient diagnostics.
Technologies that visualize multiple biomolecules at the nanometer scale in cells will enable deeper understanding of biological processes that proceed at the molecular scale. Current fluorescence-based methods for microscopy are constrained by a combination of spatial 5 resolution limitations, limited parameters per experiment, and detector systems for the wide variety of biomolecules found in cells. We present here super-resolution ion beam imaging (srIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added small molecules. Uniquely, the atomic constituents of the biomolecules themselves can often be used in our system as the "tag". We 10 visualized the subcellular localization of the chemotherapy drug cisplatin simultaneously with localization of five other nuclear structures, with further carbon elemental mapping and secondary electron visualization, down to ~30 nm lateral resolution. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. These data highlight how multiplexed super-resolution 15 techniques, such as srIBI, will enable studies of biomolecule distributions in biologically relevant subcellular microenvironments. One Sentence Summary:Three-dimensional multiplexed mass spectrometry-based imaging revealed the 20 subcellular localization of proteins and small molecules at super-resolution.3 Main Text:Form drives function in cells and tissues. This is especially true when researchers attempt deciphering of the complex interplay of molecular components that drive the biology of cells.Currently, biochemical approaches decipher meaning and mechanism from inferential experiments 5 that allow researchers to abstract a picture of how the molecular machines of a cell function. This begs the question-would it not be more useful to image these machines in their natural contexts?Fluorescence microscopy is currently the de-facto choice for biomolecular imaging, although multiplexing is often challenging due to the spectral overlap of fluorophores. Recent advancements partially overcome this limitation by using cyclic-hybridization protocols to enable multi-10 parameter visualization of proteins, RNA, and DNA (1-4). These techniques allow a range of studies from single-cells at super-resolution (5) to whole tissues (6) and have revealed a rich interplay between nucleic acids (5,7, 8). There remain challenges however, such as acquisition time for imaging and sample positional shifts between cycles, which become more pronounced at super-resolution scales (9). In addition, imaging of small molecules, such as drugs, metabolites, or 15 lipids, is also complicated since tags, such as fluorophores conjugated to biomolecules, can affect biological activities (10). The ability to directly image atomic components as labels would, with higher resolution, allow for a three-dimensional (3D) reconstruction of the distribution of mu...
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