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Dissecting tumour pathophysiology using intravital microscopy

Key Points

  • A solid tumour is an organ-like structure that consists of cancer cells and host stromal cells embedded in an extracellular matrix and nourished by a vascular network. Intravital microscopy (IVM) has provided unprecedented molecular, anatomical and functional insights into the inner workings of this organ and provided a means for testing the efficacy of various therapies.

  • IVM is a powerful optical imaging technique that allows continuous non-invasive monitoring of molecular and cellular processes in intact living tissue with 1–10 μm resolution. Such resolution is currently not possible with non-optical techniques.

  • IVM requires an appropriate animal model, a molecular probe (usually fluorescent), a microscope equipped with a digital camera detection system, an image acquisition system, and a computer to process and analyse the data to extract parameters of interest.

  • Molecular imaging has revealed heterogeneity in the tumour microenvironment (for example, pO2 and pH) and the delivery of therapeutics (micro-pharmacokinetics). The most celebrated applications of molecular imaging include measurement of promoter activity, enzyme activity and gene expression in vivo.

  • Cellular imaging has uncovered key steps in the spread of cancer from one site to the next (invasion and metastasis), and barriers to various cell-based therapies (for example, immunotherapy and gene therapy). Cellular imaging has also allowed measurement of interactions among various subpopulations of cells in a tumour.

  • Anatomical imaging has allowed quantification of morphological abnormalities in tumour vessels, as well as the size of pores in their walls.

  • Functional imaging has revealed that tumour blood flow, vascular permeability, interstitial diffusion, convection and binding are spatially and temporally heterogeneous in tumours, depend on the host–tumour interactions, and change during the course of treatment. Functional lymphatics are present only in the tumour margin and the peri-tumour tissue.

  • IVM has revealed that certain direct and indirect anti-angiogenic treatments can 'normalize' the abnormal tumour vessels so that they become more efficient. This finding underscores the importance of optimal dose and scheduling in combination therapy.

  • With the availability of hand-held microscopes and safe (Food and Drug Administration approved) molecular probes, IVM has the potential to become a useful clinical tool to monitor integrative pathophysiology and the response of optically accessible tumours to various therapies in cancer patients.

Abstract

For a systemically administered therapeutic agent to reach neoplastic cells, it must enter the blood circulation, cross the vessel wall, move through the extracellular matrix and avoid getting cleared by the lymphatics. In tumours, each of these barriers is abnormal, changes with space and time, and depends on host–tumour interactions. Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insights into these barriers and has revealed new approaches to improved detection and treatment.

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Figure 1: Four requirements for intravital microscopy.
Figure 2: Molecular imaging.
Figure 3: Cellular imaging.
Figure 4: Anatomical imaging.
Figure 5: Functional imaging.
Figure 6: Imaging therapeutic responses.

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Acknowledgements

We thank E. Brown, M. Dewhirst, M. Intaglietta, B. Seed, P. So and R. Weissleder for their helpful comments. This work was supported by National Institute of Health grants.

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DATABASES

LocusLink

bFGF

cathepsin B

CD31

CD105

ERBB2

E-selectin

HIF1α

ICAM1

PlGF

TNF-α

VCAM1

VEGF

VEGFC

VEGFR1

VEGFR2

Medscape DrugInfo

adriamycin

Herceptin

FURTHER INFORMATION

Edwin L. Steele Laboratory

Glossary

TRANS-ILLUMINATION

Microscope configuration in which the light passes from the illuminating condenser through the tissue and then into the objective lens. A green filter is often used to increase the contrast of haemoglobin. Applicable only for imaging in relatively thin tissues.

EPI-ILLUMINATION

Microscope configuration in which light comes from the objective lens to the tissue and is then collected by the same objective lens. This is useful for both fluorescent and bright-field (reflectance) microscopy. With the use of appropriate molecular probes and optical filters, specific molecular, cellular, anatomical and functional events can be detected.

CONFOCAL LASER-SCANNING MICROSCOPE

(CLSM). A high spatial-resolution fluorescence microscope that uses scanning laser light for excitation and a pinhole in the emission light path that detects a signal only from the focal plane. CLSMs can provide 1-μm three-dimensional resolution.

MULTI-PHOTON LASER-SCANNING MICROSCOPE

(MPLSM). A high spatial-resolution fluorescence microscope using low energy (long wavelength) photons that are produced by an infrared laser. Multi-photon excitation occurs only in a small volume in which the photons are focused. The long-wavelength photons and high signal-to-noise ratio allow subsurface imaging (700 μm) with 1-μm three-dimensional resolution, minimal photo-damage and longer probe lifetimes in thick samples.

FRACTAL ANALYSIS

A mathematical analysis for characterizing complex, repeating geometrical patterns at various scale lengths.

MICRO-LYMPHANGIOGRAPHY

An imaging technique to visualize functional lymphatic micro-vessels in vivo. Locally injected fluorescent macromolecules (such as fluorescein-isothiocyanate-conjugated dextran) are taken up by the lymphatics and can be visualized by intravital microscopy.

PORE CUT-OFF SIZE

Maximum size of particle that can cross the blood-vessel wall. In general, tumour vessels have a significantly larger pore cut-off size compared with their normal counterparts. Pore size depends on the host–tumour interactions and can change during tumour growth and response to therapy.

VASCULAR PERMEABILITY

A measure of the propensity of a molecule to extravasate from the vessel lumen to the tissue. Transport of systemically injected tracers, such as tetramethyl-rhodamine-labelled albumin, is monitored by intravital microscopy to estimate vascular permeability. Tumour vessels generally have high vascular permeability.

FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

(FRAP). An imaging technique to measure diffusion, convection and binding of fluorescent molecules. First, tissues are loaded with fluorescent molecules (typically fluorescein isothiocyanate labelled); strong laser light bleaches a pattern in the tissue and the recovery of fluorescence in the bleached pattern is monitored. The rate of recovery is proportional to diffusivity of the molecules in the tissue.

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Jain, R., Munn, L. & Fukumura, D. Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer 2, 266–276 (2002). https://doi.org/10.1038/nrc778

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