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  • Review Article
  • Published:

Imaging hallmarks of cancer in living mice

Nature Reviews Cancer volume 14, pages 406–418 (2014)Cite this article

Subjects

Key Points

  • Our current view on cancer hallmarks is mostly based on techniques that provide a snapshot of a large population of cells, whereas intravital microscopy (IVM) provides a view on dynamic subpopulations of both tumour and stromal cells in living animals.

  • Imaging windows allow longitudinal studies on optically inaccessible tumour tissue during consecutive imaging sessions. Most imaging windows are used to study primary tumours, but some allow for the examination of metastases.

  • To acquire high-quality fluorescent images deep inside animals, multiphoton microscopes are frequently used. The advantages of multiphoton microscopes include deep tissue penetration, reduced phototoxicity and reduced bleaching, optical sectioning and reduced tissue-mediated absorption and scattering.

  • Several contrast agents and fluorescent biosensors are available to intravitally image the activation status of multiple proteins and signalling pathways in individual cells, deep inside animals.

  • IVM demonstrated a paradoxical role of the tumour vasculature by showing that, in some cases, leaky vessels can prevent the delivery of therapeutic agents owing to elevated interstitial fluid pressure but, in other cases, leaky vessels can support the delivery of drugs.

  • Intravital imaging is a powerful tool to investigate the paradoxical role of the immune system on tumour growth and could help to clarify the best approach to stimulate the immune destruction of tumours and prevent pro-tumorigenic immune effects.

  • Intravital lineage tracing can uncover the dynamic nature of cancer stem cells and provides information on the history and fate of these cells.

  • IVM has the potential to identify the targets of cancer therapeutics and validate the in vivo mode of actions of these drugs, for example, those that inhibit proliferation or promote cell death.

  • Combining fluorescence IVM techniques with various imaging and conventional biochemical techniques holds great promise to gain further insight into the molecular mechanisms that underlie the hallmarks of cancer.

Abstract

To comprehend the complexity of cancer, the biological characteristics acquired during the initiation and progression of tumours were classified as the 'hallmarks of cancer'. Intravital microscopy techniques have been developed to study individual cells that acquire these crucial traits, by visualizing tissues with cellular or subcellular resolution in living animals. In this Review, we highlight the latest intravital microscopy techniques that have been used in living animals (predominantly mice) to unravel fundamental and dynamic aspects of various hallmarks of cancer. In addition, we discuss the application of intravital microscopy techniques to cancer therapy, as well as limitations and future perspectives for these techniques.

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Figure 1: Intravital microscopy to study tumour migration and metastasis.
Figure 2: Visualizing intratumoural heterogeneity of blood flow.
Figure 3: Intravital lineage tracing using the Confetti construct to study cancer stem cell plasticity.

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Acknowledgements

The authors would like to thank the members of the van Rheenen laboratory and J. de Rooij for their critical comments and helpful discussions. The authors apologize to those whose work is not cited owing to space limitations. The authors' research is funded by a Vidi fellowship (91710330; to J.v.R.) and equipment grants (175.010.2007.00 and 834.11.002; to J.v.R.) from the Dutch organization of scientific research (NWO), a grant from the Dutch Cancer Society (KWF; HUBR 2009–4621; to J.v.R.) and a grant from the Association for International Cancer Research (AICR; 13–0297).

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  1. Cancer Genomics Netherlands-Hubrecht Institute-KNAW & University Medical Centre Utrecht, Uppsalalaan 8, Utrecht, 3584 CT, The Netherlands

    Saskia I. J. Ellenbroek & Jacco van Rheenen

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  1. Saskia I. J. Ellenbroek
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Correspondence to Jacco van Rheenen.

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PowerPoint slides

Glossary

Stroma

Supporting structures and non-malignant cells surrounding the tumour; mainly consists of the basement membrane, fibroblasts, extracellular matrix, immune cells and vasculature.

Second harmonic generation

(SHG). Certain materials convert light of exactly half the wavelength that they are illuminated with. Owing to their large numbers of aligned α-helices, collagen fibres can be visualized through this principle.

Intravasation

The process by which cells invade through the basement membrane and the endothelium to enter blood vessels.

Photoswitchable fluorophores

Fluorophores that are designed to undergo a non-reversible colour change upon illumination with a laser.

Tongue-holding device

A small metal frame that holds the tongue of a mouse in the same position relative to the lens of a microscope, so that the same imaging position can be imaged in multiple intravital imaging sessions.

Oxygen partial pressure

(PO2). The measure of the pressure of oxygen dissolved in blood.

Quantum dots

Inorganic nanocrystals that typically have a diameter of 2–8 nm. Most are composed of an inner semiconductor core of CdSe, an outer shell of ZnS and an organic coating to make the quantum dot biocompatible. Quantum dots can be engineered to emit fluorescent light in the ultraviolet to infrared spectrum by varying their size.

Interstitial fluid pressure

High interstitial fluid pressure (for example, in the range 10–20 mm Hg) is a characteristic of many human tumours. This build-up of hydrostatic pressure within tumours is thought to be partly due to abnormal and highly permeable tumour vessels and decreased lymphatic drainage.

Spinning-disk confocal microscopy

A confocal scanning microscopy technique that is optimized for imaging speed because it uses an array of moving pinholes on a disc to scan the specimen.

Adult stem cells

A rare population of cells in healthy tissue that have the ability to self-renew and differentiate into specialized cells.

Transit-amplifying cells

Cells that are derived from stem cells, which divide a finite number of times until they become differentiated.

Lineage tracing experiments

Experiments in which the expression of fluorescent markers (such as green fluorescent protein (GFP)) is genetically induced in individual cells, so that their progeny also express this marker and therefore the lineage can be followed.

Cre–Lox system

The Cre recombinase enzyme recombines Lox sequences, which results in the excision or reversion of the DNA that is flanked by Lox sites. This method is often used to activate or inactivate the expression of genes.

Mammary imaging window

A small ring with a coverslip that is surgically implanted into the skin and on top of the mammary gland to provide optical access to breast tissue during repeated intravital imaging sessions.

Ultrasound backscatter microscopy

(UBM). An imaging technique in which an ultrasound pulse is delivered into biological tissue and the backscattered ultrasound is detected. The amplitude of the scattered signal forms the image.

Photoacoustic imaging

(PAI). An imaging technique in which non-ionizing laser pulses are delivered into biological tissues and the absorbed energy leads to ultrasonic emission. The amplitude of the detected emission forms the image.

Thresholding methods

Image segmentation based on pixel intensities.

Abdominal imaging window

A small titanium ring fitted with a coverslip that is surgically implanted into the abdominal wall of an animal to provide optical access to abdominal organs such as the colon, small intestine, liver, spleen, and kidney during repeated intravital imaging sessions.

Confocal laser endomicroscopy

An emerging endoscopic technology that permits high-resolution imaging of the intestine at the cellular or subcellular level.

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Ellenbroek, S., van Rheenen, J. Imaging hallmarks of cancer in living mice. Nat Rev Cancer 14, 406–418 (2014). https://doi.org/10.1038/nrc3742

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