By Robert M. Hoffman1 About the author
Naturally fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis and angiogenesis. These multicoloured proteins have allowed the colour-coding of cancer cells growing in vivo and enabled the distinction of host from tumour with single-cell resolution. Visualization of many aspects of cancer initiation and progression in vivo should be possible with fluorescent proteins.
Fluorescent proteins can be used to visualize any type of cancer process, including primary tumour growth, tumour cell motility and invasion, metastatic seeding and colonization, angiogenesis, and the interaction between the tumour and its microenvironment (tumour–host interaction). Fluorescent proteins of many different colours have now been characterized and these can be used to colour-code cancer cells of a specific genotype or phenotype. For example, the behaviour of highly metastatic cancer cells labelled with green fluorescent protein (GFP) and low metastatic cancer cells labelled with red fluorescent protein (RFP) can be directly compared in vivo. Alternatively, the host and the tumour can be differentially labelled with fluorescent proteins — a transgenic mouse expressing GFP in all of its cells (or in specific cells such as endothelial cells) transplanted with tumour cells expressing RFP enables the interaction between the tumour cells and the host cells to be visualized in real time.
The fact that the excitation wavelengths for some fluorescent proteins are long enables real-time imaging to take place without harming the animals' tissues. Longer wavelength light causes few damaging events to proteins and DNA because of its lower energy. The long wavelength excitation of fluorescent proteins also reduces the extent of photobleaching compared with dyes that have a shorter wavelength excitation. Therefore, real-time tracking of tumour growth and metastasis can be carried out in the intact animal. For single-cell resolution, reversible acute skin-flaps as well as CHRONIC-TRANSPARENT WINDOW models can be used over many parts of the body (skin, brain, lung, liver, and so on). Real-time imaging with fluorescent proteins is especially important when evaluating the efficacy of therapeutics on metastasis and tumour recurrence.
The first use of GFP to visualize cancer cells in vivo was by Chishima. They stably transfected tumour cells with GFP and transplanted these into several mouse models, including ORTHOTOPIC models that have a high metastatic capacity. They showed that in excised live tissue, with no additional preparation, metastases could be observed in any organ at the single-cell level. In addition, cells were visualized in the process of INTRAVASATION and EXTRAVASATION. The visualization of single metastatic cells in tissue is beyond the capabilities of standard histological techniques and so such ex vivo studies enabled, for the first time, micrometastases (including dormant cells) to be visualized in unfixed or unprocessed tissue.
Intravital imaging using fluorescent proteins
Before the introduction of fluorescent proteins, in vivo imaging was limited to the study of cells that were transiently labelled with vital dyes. Stable fluorescent labelling, achieved using vectors that express fluorescent proteins, now allows the direct imaging of single cells in vivo.
Using what is termed intravital microscopy — observation of a tumour of interest either through surgically created chronic-transparent windows or directly through the opened skin of living animals — single cancer cells have been visualized. High-resolution INTRAVITAL VIDEO MICROSCOPY of GFP-expressing tumour cells provides a powerful tool for directly observing steps in the metastatic process. Individual, non-dividing cells as well as micro- and macrometastases can be clearly visualized and quantified. Cellular details, such as pseudopodial projections, can be clearly seen. Observed tumour cell motility at the single-cell level, including movement in and out of blood vessels, using GFP-expressing cells. Condeelis. have used GFP imaging to view cells in time-lapse images in a single optical section using a confocal microscope. The polarity of tumour cells, along with their response to chemotatic cytokines, has been visualized by intra-vital. These techniques enable a greater understanding of tumour cell migration in vivo.
Figure a |The image shows HT-1080 human fibrosarcoma cells migrating in a skin capillary 14 hours after injection into the heart. Histone H2B–green fluorescent protein (GFP) is evident in the nucleus, and retrovirally expressed red fluorescent protein (RFP) is seen in the cytoplasm. Note the high degree of cell and nuclear deformation of tumour cells. Image courtesy of K. Yamauchi, N. Yamamoto, P. Jiang, and R.M.H.
Figure b | This image shows an HT-1080 human fibrosarcoma cell, expressing GFP in the nucleus and RFP in the cytoplasm, extravasating from a blood vessel in the skin (indicated by the arrow). Note that the blood vessel contains numerous tumour cells. The extravasating cell was visualized 2 hours after cell injection of the labelled tumour cells into the heart. Image courtesy of K. Yamauchi, N. Yamamoto, P. Jiang and R.M.H.
Figure c | This image shows extravasated Lewis lung carcinoma cells growing within a blood vessel. These cells were labelled in the nucleus with histone H2B–GFP and in the cytoplasm with a retrovirally-expressed RFP. The dual-colour cells were injected into the epigastric cranialis vein and the image from a live mouse with a skin flap was taken 120 hours post-injection. Image courtesy of K. Yamauchi and R.M.H.
