Abstract
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.
Highlights
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 previously demonstrated the ability of secondary ion beam imaging (SIMS), a form of mass spectrometry imaging (MSI), to image multiple targetedproteins in tissue samples using multiplexed ion beam imaging (MIBI)[13]
Cells are cultured on a conductive silicon substrate and treated with cisplatin, a small molecule containing a platinum atom that can be efficiently ionized by the cesium beam
Summary
The 19F signal overlapped with nuclear regions low in 31P confirming the specificity of antiH3K27Ac-19F/FITC (Fig. S5) These results demonstrate that detection of MoC-Abs labeled with 19F by ion beam imaging can reveal subnuclear structures. We performed 3D rendering of 400 planes from a single HeLa cell labeled with anti-nucleolin-19F, anti-H3K9me381Br, anti-H3K27Ac-127I, and anti-SC35 (recognized by streptavidin-197Au), creating a composite 3D visualization of the nucleus (Fig. 3d and Movie S3) These results indicate how HD-MIBI enables whole-cell reconstruction of the genome and epigenome. We colored the voxels in each nucleus based on these groups and projected them onto a 2D plane to better visualize the spatial distribution of the nuclear neighborhoods (Fig. 4d) These images reveal distinctive nuclear structures, such as nucleoli (neighborhood 1), nuclear speckles (neighborhood 4), and chromatin that resembles lamin-associated domains (neighborhood 8). These results support a model wherein selective depletion of cisplatin from the nucleus in cells enables them to survive combination platinum-based therapy and bromodomain inhibition
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