Green fluorescent protein imaging of tumour growth, metastasis, and angiogenesis in mouse models

doi:10.1016/S1470-2045(02)00848-3

The Lancet Oncology

Volume 3, Issue 9, September 2002, Pages 546-556

https://doi.org/10.1016/S1470-2045(02)00848-3 Get rights and content

Summary

We have developed a way of imaging metastases in mice by use of tumour cells expressing green fluorescent protein (GFP) that can be used to examine fresh tissue, both in situ and externally. These mice present many new possibilities for research including real-time studies of tumour progression, metastasis, and drug-response evaluations. We have now also introduced the GFP gene, cloned from bioluminescent organisms, into a series of human and rodent cancer-cell lines in vitro, which stably express GFP after transplantation to rodents with metastatic cancer. Techniques were also developed for transduction of tumours by GFP in vivo. With this fluorescent tool, single cells from tumours and metastases can be imaged. GFP-expressing tumours of the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs have also been visualised externally by use of quantitative transcutaneous whole-body fluorescence imaging. GFP technology has also been used for real-time imaging and quantification of angiogenesis.

Section snippets

Green fluorescent protein

In order to externally image and follow the natural course or impediment of tumour progression and metastasis, high specificity, a strong signal, high resolution, and good physiological conditions are necessary. The GFP gene, cloned from the bioluminescent jellyfish Aequorea victoria,¹⁰ was chosen to satisfy these conditions since it has great potential for use as a cellular marker.11, 12 GFP cDNA encodes a 283 aminoacid monomeric polypeptide with a molecular weight of 27 kDa13, 14 that

Stable transduction of tumour cells with GFP

Several groups have selected tumour cell lines to stably express GFP at high levels both in vitro and in vivo. These cells can be transplanted into animals and visualised in situ in fresh tissues (figure 1).24, 25, 26, 27, 28 Furthermore, tumour cells expressing GFP have been visualised with or without subsequent colonisation in all the major organs including liver, lung, brain, spinal cord, axial skeleton, and lymph nodes.24, 28 GFP models of metastatic disease have been developed for lung

Ovarian cancer

Studies in which GFP-expressing Chinese hamster ovary tumour fragments (CHO-K1-GFP) of about 1 mm³ were implanted into the ovarian serosa of nude mice by surgical orthotopic implantation (SOI) resulted in the development of ovarian tumours.26, 35 The tumours, which were strongly fluorescent, subsequently spread throughout the peritoneal cavity, including the colon, cecum, small intestine, spleen, and peritoneal wall. GFP fluorescence was used to track tumour spread; numerous micrometastases

Intravital imaging of GFP-expressing cells

Intravital videomicroscopy can be used to visualise sequential steps in metastasis by use of CHO-K1 cells that stably express GFP. In mouse liver, the stages from the initial arrest of cells in the microvasculature up to the growth and angiogenesis of metastases have been recorded.⁵ Individual non-dividing cells, as well as micro and macrometastases were visualised and quantified; additional cellular details such as pseudopodial projections were also detected. Micrometastases were found to

Whole-body imaging of tumour growth and metastasis

External whole-body imaging of mice with primary and metastatic tumours that are genetically labelled with the fluorescent proteins GFP and RFP is a simple but powerful tool for investigating tumour development. The technology is based on the bright intrinsic fluorscence of GFP and RFP, which is partly caused by the high quantum yield of these fluorophores.19, 23 For tumour cells to be visualised with this technique, they must be transduced with GFP or RFP genes, such that they become brightly

Whole-body imaging of metastatic lesions

Transplanted mice with metastatic lesions of GFP-expressing tumours in the colon, brain (figure 2), liver, lymph nodes, and bone (figure 3) have been used to produce images of metastasis. These images are used for real-time quantitative measurement of primary and metastatic tumour growth for each of these organs. GFP-expressing cells emit a bright fluorescence signal compared with background fluorescence from other tissue. The signal is so strong and selective that external images of

Whole-body and intravital imaging of angiogenesis and intravascular tumour cells

Tumour angiogenesis can also be visualised by use of GFP techniques. The footpads of mice are quite transparent with few resident blood vessels and are therefore ideal for quantitative imaging of tumour angiogenesis in intact animals. Vessels can be seen because of their striking contrast to the GFP fluorescence of the tumour tissue.⁴⁸ Yang and colleauges injected GFP-expressing Lewis lung carcinoma cells subcutaneously into the footpad of nude mice and, using whole-body imaging, they found

Imaging GFP tumour cells in blood vessels

Following injection of tumour cells stably expressing GFP in to the tail vein of mice, it was possible to visualise single tumour cells in blood vessels.²⁶ With intravital microscopy, Naumov and colleagues visualised GFP tumour cells colonising various organs after extravasation.⁵ Huang,⁵⁵ Li,⁵⁶ and their respective co-workers visualised GFP tumour-cell-vessel interaction by use of skin window chambers in rodents and observed angiogenic effects very early in tumour colony formation. In an

GFP labeling of VEGF in vivo

Brown and colleagues³ showed that multiphoton laser-scanning microscopy could provide high resolution three-dimensional images of angiogenesis gene expression and that this techniques could be used to investigate deeper regions of GFP-expressing tumours in dorsal skin-fold chambers. To monitor the activity of the vascular endothelial growth factor (VEGF) promoter, Fukumura and colleagues made transgenic mice that express GFP under control of the VEGF promoter.60, 61 Multiphoton laser scanning

Use of GFP to monitor potential expression of heat-shock proteins in vivo

Mouse melanoma cells were stably transfected with a plasmid containing the GFP gene linked to a heat shock protein gene promoter, which reacts to physiological stress.⁶³ At physiological temperature, GFP expression in experimental mouse tumours was undetectable. However, the tumour cells started to fluoresce brightly in response to an increase in temperature (heat shock) both in vitro and in vivo. Thus, GFP could be a useful marker for studies of mammalian heat shock proteins.

Clinically applicable models of GFP tumour imaging

Several studies have focused on delivering the GFP gene selectively to tumours in order to provide a marker for the development of new metastases. Hasegawa and colleagues administered the GFP gene to nude mice with human stomach tumours growing intraperitoneally, in order to visualise future regional and distant metastases.⁶⁴ GFP retroviral supernatants were injected intraperitoneally from day 4 to day 10 following implantation of the cancer cells. A laparotomy was done every other week so that

Fluorescent reporter gene for human T cells

Normal, human, peripheral-blood T lymphocytes were transduced with a retroviral HSV-TK-GFP (vGFPTKfus) and nucleus-restricted green fluorescence was observed. Sorting allowed for selection of GFP-expressing T lymphocytes. The ability to target GFP-expressing T lymphocytes to tumours could have many clinical uses.⁶⁹

Bone-marrow protection by transfer of drug-resistance genes coupled to GFP

A retroviral vector expressing human O6-methylguanine-DNA methyltransferase (MGMT) and GFP was developed for stem-cell protection in a murine transplant model. Mice transplanted with transduced cells showed significant resistance to the myelosuppressive effects of temozolomide, a DNA-methylating drug, and O₆-benzylguanine, a drug that inhibits MGMT. Following drug treatment, increases in GFP-positive peripheral blood cells were seen. Secondary transplant experiments proved that selection had

A senescence programme controlled by p53 and p16^INK4a affects chemotherapy

The GFP primary lymphomas derived from Eμ-myc transgenic mice respond to chemotherapy by undergoing apoptosis and engaging a premature senescence programme controlled by p53 and p16^INK4a. Therefore, tumours respond poorly to cyclophosphamide therapy if their p53 or INK4a/ARF genes are disrupted; this can be seen with GFP whole-body imaging. It has also been shown that mice bearing tumours capable of drug-induced senescence have a much better prognosis after chemotherapy than those harboring

Conclusions

Tumour cells stably expressing GFP in vivo are a powerful new tool for cancer research. Stability of expression has been studied by Naumov and colleagues⁵ who noted that all the CHO-K1-GFP cells used in their study were stably fluorescent (measured by flow cytometry) even after 24 days of growing in medium where they were deprived of selective pressure. This finding implies that GFP can be stably expressed in cells in vivo. This feature has proved true for all cells studied so far and is

Search strategy and selection criteria

References for this review were identified from the results of searches of PubMed and selected from the references of relevant articles. The search terms used were ~green fluorescent protein”, “cancer”, and ~in vivo”. Searches were limited to papers published in English between 1997 and 2002 that discussed imaging investigations.

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ReviewGreen fluorescent protein imaging of tumour growth, metastasis, and angiogenesis in mouse models

Summary

Section snippets

Green fluorescent protein

Stable transduction of tumour cells with GFP

Ovarian cancer

Intravital imaging of GFP-expressing cells

Whole-body imaging of tumour growth and metastasis

Whole-body imaging of metastatic lesions

Whole-body and intravital imaging of angiogenesis and intravascular tumour cells

Imaging GFP tumour cells in blood vessels

GFP labeling of VEGF in vivo

Use of GFP to monitor potential expression of heat-shock proteins in vivo

Clinically applicable models of GFP tumour imaging

Fluorescent reporter gene for human T cells

Bone-marrow protection by transfer of drug-resistance genes coupled to GFP

A senescence programme controlled by p53 and p16INK4a affects chemotherapy

Conclusions

Search strategy and selection criteria

J Surg Res

Neoplasia

Gene

Gene

FEBS Lett

Am J Pathol

Gynecol Oncol

J Neurosci Methods

Microvasc Res

Cell

Mol Ther

Cancer Cell

Cell

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Nat Med

Bacterial lacZ gene as a highly sensitive marker to detect micrometastasis formation during tumor progression.

Cancer Res

In vivo measurement of gene expression, angiogenesis and physicological function in tumors using multiphoton laser scanning microscopy.

Nat Med

Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.

J Cell Sci

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.

Nat Biotechnol

In vivo molecular target assessment of matrix metalloproteinase inhibition.

Nat Med

Visualizing the kinetics of tumor-cell clearance in living animals.

Proc Natl Acad Sci USA

Green fluorescent protein as a marker for gene expression.

Science

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.

Nat Biotechnol

Chemical structure of the hexapeptide chromophore of the Aequorea green fluorescent protein.

Biochemistry

The molecular structure of green fluorescent protein.

Nat Biotechnol

Energy transfer in a bioluminescent system.

J Cell Physiol

Improved green fluorescent protein by molecular evolution using DNA shuffling.

Nat Biotechnol

Red-shifted excitation mutants of the green fluorescent protein.

Biotechnology

Improved green fluorescence.

Nature

A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells.

J Virol

The structure of the chromophore within DsRed, a red fluorescent protein from coral.

Proc Natl Acad Sci USA

Fluorescent proteins from nonbioluminescent Anthozoa species.

Nat Biotechnol

Governing step of metastasis visualized in vitro.

Proc Natl Acad Sci USA

Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression.

Cancer Res

Metastatic patterns of lung cancer visualized live and in process by green fluorescent protein expression.

Clin Exp Metastasis

Visualization of the metastatic process by green fluorescent protein expression.

Anticancer Res

Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression.

Cancer Res

A model of spontaneous lung metastases visualised in fresh host tissue by green fluorescent protein expression.

Clin Exp Metastasis

Review
Green fluorescent protein imaging of tumour growth, metastasis, and angiogenesis in mouse models

A senescence programme controlled by p53 and p16^INK4a affects chemotherapy