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Imaging of anticancer drug action in single cells

  • Review Article
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From Nature Reviews Cancer

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Key Points

  • The in vivo microscopy toolbox enables extended live-animal 3D imaging in orthotopic disease sites, deeper imaging through tissue and multiple simultaneous molecular measurements.

  • Imaging of multiple single cells can assess tumour heterogeneity, tumour evolution following drug treatment and drug–target binding; it also enables visualization of micro-anatomical structures and distinct and rare cell types.

  • Fluorescently labelled drugs and nanoformulations reveal mechanisms of delivery and binding within tumours.

  • Imaging can show differences in drug response in vivo compared with in vitro tissue culture models.

  • Components of the tumour microenvironment, including the extracellular matrix, immune cells and vasculature, can be studied for their role in influencing drug action.

  • The distribution and effect of immunotherapies including those that block immune checkpoints can be visualized in the tumour and draining lymph nodes.

  • In vivo microscopy provides a complementary high-resolution perspective to lower-resolution imaging modalities such as positron emission tomography–computed tomography and magnetic resonance imaging that are used in the clinic.

Abstract

Imaging is widely used in anticancer drug development, typically for whole-body tracking of labelled drugs to different organs or to assess drug efficacy through volumetric measurements. However, increasing attention has been drawn to pharmacology at the single-cell level. Diverse cell types, including cancer-associated immune cells, physicochemical features of the tumour microenvironment and heterogeneous cell behaviour all affect drug delivery, response and resistance. This Review summarizes developments in the imaging of in vivo anticancer drug action, with a focus on microscopy approaches at the single-cell level and translational lessons for the clinic.

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Figure 1: Imaging pharmacokinetic/pharmacodynamic processes with in vivo microscopy.
Figure 2: Direct and indirect modes of pharmacokinetic imaging.
Figure 3: Example drug delivery problems elucidated by microscopy.
Figure 4: Imaging drug binding and problems thereof.
Figure 5: Imaging the role of myeloid cells in influencing drug action.

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Acknowledgements

The authors thank M. Pittet (Massachusetts General Hospital (MGH)), C. Vinegoni (MGH) and T. Mitchison (Harvard Medical School) for thoughtful review, and R. Kohler (MGH) in the Weissleder lab for images and movies. This work was partly funded by NIH grants R01EB010011, R01HL122208, P50GM107618, T32CA079443 and K99CA207744.

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  1. Center for Systems Biology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Miles A. Miller & Ralph Weissleder

  2. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Ralph Weissleder

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Correspondence to Ralph Weissleder.

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Supplementary information

Supplementary information S1 (table)

Useful fluorescent reporters for imaging cell types, cellular pathways, and physiological features. (PDF 211 kb)

Supplementary information S2 (table)

Key and recent references describing the optical imaging techniques compared in Figure 1. (PDF 202 kb)

Tumour-associated myeloid cells and nanotherapeutic extravasation.

Stroma adjacent to an implanted syngeneic lung cancer tumour was imaged in a dorsal window chamber, using a chemokine (C-X3-C motif) receptor 1 (Cx3cr1)GFP/+ reporter mouse model. Multicolour imaging reveals migration and perivascular localization of GFP+ monocytes, TAMs, and dendritic cells. Also shown are vessel perfusion and extravasation kinetics of a model therapeutic nanoformulation based on polymeric micelles9, which was intravenously injected at the start of the movie. These data highlight relatively low levels of nanoparticles extravasating from the vessels into the tissue, indicated by lack of extravascular nanoparticle accumulation throughout most of the sequence. As an exception, several transient bursts of vessel leakage (marked by arrows pointing to sites where nanoparticles escape vessels and are transported into tissue), which occur near perivascular immune cells are shown towards the end of the movie. These representative data are based on experiments similar to those previously published1 (Weissleder lab). Bursts of vascular extravasation can be amplified with adjuvant treatments, such as local conformal irradiation, to enhance nanotherapeutic delivery to solid tumours10. The mechanisms and impact of bursting on nanotherapeutic delivery are extensively described elsewhere10. (MOV 8170 kb)

Olaparib-CID PK/PD. Time lapse imaging enables kinetic measurements to be made through a dorsal window chamber.

This movie shows extravasation, cellular uptake, and nuclear retention of the fluorescently tagged PARP inhibitor, olaparib (green)39, in fibrosarcoma tumour cells (HT1080 cells) that express the histone H2B– mApple fusion protein (red). This representative movie is based on data similar to those previously published39 (Weissleder lab). (MOV 5555 kb)

Imaging cell-cycle and mitotic defects.

Combined imaging of the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) cell cycle reporter and histone H2B provides simultaneous visualization of cell migration, cell-cycle phase, and mitotic defects, for instance, related to metaphase arrest and chromosomal mis-segregation following treatment with a microtubule-targeting drug (paclitaxel). Lectin reveals microvasculature structure. This representative movie is based on data similar to those previously published97 (Weissleder lab). (MOV 9856 kb)

Glossary

Pharmacokinetics

(PK). The collective interactions of a drug with an organism that determine the fate of the former through processes including absorption, distribution, metabolism and excretion (ADME).

Pharmacodynamics

(PD). The effects of a drug on an organism, defined broadly here to include processes of drug–target binding, as well as corresponding changes in downstream biochemical pathways, cellular phenotypes and disease progression.

Organ clearing

Solvent-based processing of excised organs for removal of light-scattering material, such as lipids and haem, thus improving optical imaging deep through tissue.

Tissue expansion

A process by which swelling polymer gel is synthesized in excised tissue specimens, causing structural expansion and enabling molecular features to be resolved at greater apparent resolution.

Multiplexed histology by image cycling

Immunostaining of tissue sections is multiplexed to image multiple markers using multicolour labelling and image cycling, which involves repeated immunostaining, imaging, stain removal and restaining of different markers.

Volumetric IVM

A common technique used in intravital microscopy (IVM), whereby confocal or multiphoton imaging at multiple planes of focal depth through tissue is reconstructed into images of 3D tissue volumes.

Image segmentation

A post-processing technique, typically performed by algorithmic classification of shapes and intensities, to identify and outline biologically relevant compartments such as cells and vessels.

Coherent anti-Stokes Raman spectroscopy (CARS)

A technique for imaging molecular vibrational signatures as with Raman spectroscopy, but using multiple photons to produce a coherent optical signal with emitted photons of higher energy than the individual absorbed photons (that is, an anti-Stokes signal). In biological applications, CARS is useful for visualizing lipid-rich material such as myelin in a label-free manner.

Image registration

The map** of multiple images, typically acquired from different imaging modalities (for example, positron emission tomography and computed tomography) or acquisition settings, onto a shared spatial coordinate system for integrated analysis.

Syngeneic

Tumours that share sufficiently similar genetic background with the host that they can be implanted into immunocompetent animals without provoking immune rejection.

Fiducial markers

An image feature or set of features, such as defined objects placed in the field of view, that are used as points of reference to orient or register the image, to identify structural or cellular elements, to stabilize the field of view and to correct for distortions.

Mass spectrometry imaging

This technique scans a tissue with a locally ionizing beam, thereby ejecting molecules for mass spectrometry analysis and allowing multiplexed quantification of biological molecules, drugs and metal-labelled antibodies.

Extravasation kinetics

The rates at which small molecules, proteins, nanoparticles and cells move from the vasculature into neighbouring tissue.

Autochthonous

Describes tumours that arise in the host animal without surgical cell implantation. These typically form spontaneously from engineered conditional expression of oncogenic mutations in particular tissue sites.

Enhanced permeability and retention (EPR) effects

The collective influence of the multiple factors, such as increased vascular permeability, dysfunctional lymphatics and increased immune-cell infiltration, that cause macromolecules and nanoparticles to passively accumulate in some solid tumours.

Fluorescence anisotropy

The property of a fluorochrome-emitting light with biased polarization, which, for instance, can be the result of excitation with a polarized light source, combined with a slow rate of depolarization of emitted photons, owing to the binding of a fluorescent drug conjugate with its macromolecular protein target.

Quantum-dot beacons

Fluorescent semiconductor nanoparticles with high brightness, photostability and narrow yet tunable fluorescence spectra that have been conjugated to antibodies for robust multicolour imaging.

Paradoxical activation

Inhibitor response that is the opposite of what is expected based on the drug's putative mechanism of action. This generally occurs when drug treatment upregulates the target pathway, for instance, as noted with BRAF inhibitors that can stimulate MAPK activity in cells with wild-type BRAF.

Fluorescence recovery after photobleaching

(FRAP). The fluorescence microscopy technique of locally photobleaching a tissue or cell section and monitoring the rate at which fluorescence returns, which typically reveals transport kinetics such as molecular diffusion within a cell plasma membrane.

Neutrophil extracellular traps

(NETs). Networks of fibrillar extracellular material produced by neutrophils to capture and contain pathogens, which also have a role in cancer metastasis, thrombosis and inflammation. NETs primarily consist of neutrophil-derived DNA, granule proteins and chromatin.

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Miller, M., Weissleder, R. Imaging of anticancer drug action in single cells. Nat Rev Cancer 17, 399–414 (2017). https://doi.org/10.1038/nrc.2017.41

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