Tumour cells can be stably transfected with fluorescent proteins.
Tumours and metastases that express fluorescent proteins can be visualized non-invasively in intact animals.
Transgenic mice can express a fluorescent protein in all cells or in specific cells, depending on the linkage of the fluorescent protein. These mice can be transplanted with tumours expressing different-coloured fluorescent proteins to create a dual-colour image of the tumour–host interaction.
Tumour cells can express two or more different-coloured fluorescent proteins. For example, the nucleus can be labelled with green fluorescent protein and the cytoplasm with red fluorescent protein. This enables nuclear–cytoplasmic dynamics to be visualized in vivo.
In vivo imaging can be at the single-cell level. For single-cell imaging on deep organs, reversible skin-flaps and chronic windows can be used. Single-cell imaging can be used to study cancer cell invasion, seeding in distant organs and dormancy.
Fluorescent protein imaging has significant advantages over luciferase imaging, including brighter signals, substrate independence, availability in multiple colours, and simpler and cheaper equipment requirements.
In vivo fluorescent imaging can be used to visualize the efficacy of candidate cancer drugs in real time in mouse models of human cancer.
Fluorescent proteins can be used for 'molecular imaging' to visualize the effects of single-gene changes — for example, on cancer metastasis or drug sensitivity.
Future uses of fluorescent proteins in human cancer diagnosis and therapy are possible — for example, in mouse models, tumours can be selectively and stably transformed in vivo by viral vectors. In the future such an approach might be used in humans to visualize tumour growth and response to therapy in real time.
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.
Subscribe to Journal
Get full journal access for 1 year
only $22.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Yang, M. et al. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc. Natl Acad. Sci. USA 97, 1206–1211 (2000). The first paper to demonstrate whole-body imaging using GFP-expressing tumours.
Yang, M. et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model. Proc. Natl Acad. Sci. USA 99, 3824–3829 (2002).
Yamamoto, N. et al. Real-time imaging of individual color-coded metastatic colonies in vivo. Clin. Exp. Metastasis 20, 633–638 (2003).
Brown, E. B. et al. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nature Med. 7, 864–868 (2001).
Katz, M. H. et al. Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer. Cancer Res. 64, 1828–1833 (2004).
Chishima, T. et al. Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res. 57, 2042–2047 (1997). The first paper to use GFP to visualize cancer cells in vivo.
Jain, R. K., Munn, L. L. & Fukumura, D. Dissecting tumour pathopysiology using intravital microscopy. Nature Rev. Cancer 2, 266–276 (2002).
Naumov, G. N. et al. Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J. Cell Sci. 112, 1835–1842 (1999).
Farina, K. L. et al. Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Res. 58, 2528–2532 (1998).
Condeelis, J. & Segall, J. E. Intravital imaging of cell movement in tumours. Nature Rev. Cancer 3, 921–930 (2003).
Chishima, T. et al. Metastatic patterns of lung cancer visualized live and in process by green fluorescent protein expression. Clin. Exp. Metastasis 15, 547–552 (1997).
Huang, M. S. et al. Establishment of fluorescent lung carcinoma metastasis model and its real-time microscopic detection in SCID mice. Clin. Exp. Metastasis 19, 359–368 (2002).
Li, C. Y. et al. Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models. J. Natl Cancer Inst. 92, 143–147 (2000).
Moore, A., Marecos, E., Simonova, M., Weissleder, R. & Bogdanov, A. Jr. Novel gliosarcoma cell line expressing green fluorescent protein: a model for quantitative assessment of angiogenesis. Microvasc. Res. 56, 145–153 (1998).
Al-Mehdi, A. B. et al. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nature Med. 6, 100–102 (2000).
Wong, C. W. et al. Intravascular location of breast cancer cells after spontaneous metastasis to the lung. Am. J. Path. 161, 749–753 (2002).
Yamamoto, N. et al. Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression. Cancer Res. 64, 4251–4256 (2004). First paper to use dual-colour cancer cells with GFP in the nucleus and RFP in the cytoplasm to visualize nuclear and cellular dynamics in vivo.
Yamauchi, K. et al. Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration. Cancer Res. 65, 4246–4252 (2005)
Chang, Y. S. et al. Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc. Natl Acad. Sci. USA 97, 14608–14613 (2000).
Li, L. et al. Nestin expression in hair follicle sheath progenitor cells. Proc. Natl Acad. Sci. USA 100, 9958–9961 (2003).
Amoh, Y. et al. Nascent blood vessels in the skin arise from nestin-expressing hair follicle cells. Proc. Natl Acad. Sci. USA 101, 13291–13295 (2004).
Amoh, Y. et al. Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by doxorubicin. Cancer Res. 65, 2337–2343 (2005).
Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Fukumura, D., Yuan, F., Monsky, W. L., Chen, Y. & Jain, R. K. Effect of host microenvironment on the microcirculation of human colon adenocacinoma. Am. J. Pathol. 151, 679–688 (1997).
Fukumura, D. et al. Tumor induction of VEGF promoter activity in stromal cells. Cell 94, 715–725 (1998).
Harms, J. F. & Welch, D. R. MDA-MB-435 human breast carcinoma metastasis to bone. Clin. Exp. Metastasis 20, 327–334 (2003).
Ito, S. et al. Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line. Int. J. Cancer 93, 212–217 (2001).
Mook, O. R. et al. Visualization of early events in tumor formation of eGFP-transfected rat colon cancer cells in liver. Hepatology 38, 295–304 (2003).
Sturm, J. W. et al. Enhanced green fluorescent protein-transfection of murine colon carcinoma cells: key for early tumor detection and quantification. Clin. Exp. Metastasis 20, 395–405 (2003).
Wong, C. W. et al. Apoptosis: an early event in metastatic inefficiency. Cancer Res. 61, 333–338 (2001).
Yamamoto, N. et al. Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging. Cancer Res. 63, 7785–7790 (2003).
Glinskii, A. B. et al. Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models. Cancer Res. 63, 4239–4243 (2003).
Berezovskaya, O. et al. Increased expression of apoptosis inhibitor protein XIAP contributes to anoikis resistance of circulating human prostate cancer metastasis precursor cells. Cancer Res. 65, 2378–2386 (2005).
Paris, S. et al. Inhibition of tumor growth and metastatic spreading by overexpression of inter-α-trypsin inhibitor family chains. Int. J. Cancer 97, 615–620 (2002).
Almgren, M. A., Henriksson, K. C., Fujimoto, J. & Chang, C. L. Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma. Mol. Cancer Res. 2, 387–394 (2004).
Goldberg, S. F., Harms, J. F., Quon, K. & Welch, D. R. Metastasis-suppressed C8161 melanoma cells arrest in lung but fail to proliferate. Clin. Exp. Metastasis 17, 601–607 (1999).
Goodison, S. et al. Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from non-metastatic breast tumors as revealed by labeling with green fluorescent protein. Clin. Cancer Res. 9, 3808–3814 (2003).
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997). First development of a mouse with GFP in essentially all cells.
Yang, M. et al. Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells. Proc. Natl Acad. Sci. USA 100, 14259–14262 (2003). First dual-colour tumour–host model. The model uses RFP-tumours transplanted into GFP mice.
Duda, D. G. et al. Differential transplantability of tumor-associated stromal cells. Cancer Res. 64, 5920–5924 (2004).
Sweeney, T. J. et al. Visualizing the kinetics of tumor-cell clearance in living animals. Proc. Natl Acad. Sci. USA 96, 12044–12049 (1999).
Contag, C. H., Jenkins, D., Contag, P. R. & Negrin, R. S. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2, 41–52 (2000).
Burgos, J. S. et al. Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice. Biotechniques 34, 1184–1188 (2003).
Morin, J. & Hastings, J. Energy transfer in a bioluminescent system. J. Cell. Physiol. 77, 313–318 (1971).
Cormack, B., Valdivia, R. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).
Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14, 315–319 (1996).
Delagrave, S., Hawtin, R. E., Silva, C. M., Yang, M. M. & Youvan, D. C. Red-shifted excitation mutants of the green fluorescent protein. Biotechnology 13, 151–154 (1995).
Heim, R., Cubitt, A. B., Tsien, R. Y. Improved green fluorescence. Nature 373, 663–664 (1995). References 47 and 48 were the first papers to generate GFP mutants in the chromophore for increased fluorescence as well as new colours.
Zolotukhin, S., Potter, M., Hauswirth, W. W., Guy, J. & Muzyczka, N. A 'humanized' green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70, 4646–4654 (1996).
Cody, C. W., Prasher, D. C., Welstler, W. M., Prendergast, F. G. & Ward, W. W. Chemical structure of the hexapeptide chromophore of the Aequorea green fluorescent protein. Biochemistry 32, 1212–1218 (1993). First paper to demonstrate the chromophore aminoacid sequence in GFP. This paper enabled later studies to generate brighter mutants and new colours.
Ray, P., De, A., Min, J. J., Tsien, R. Y. & Gambhir, S. S. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 64, 1323–1330 (2004).
Yang, M., Baranov, E., Moossa, A. R., Penman, S. & Hoffman, R. M. Visualizing gene expression by whole-body fluorescence imaging. Proc. Natl Acad. Sci. USA 97, 12278–12282 (2000).
Yang, M. et al. Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors. Proc. Natl Acad. Sci. USA 98, 2616–2621 (2001).
Katz, M. H. et al. A novel red fluorescent protein orthotopic pancreatic cancer model for the preclinical evaluation of chemotherapeutics. J. Surg. Res. 113, 151–160 (2003).
Katz, M. H. et al. Selective antimetastatic activity of cytosine analog CS-682 in a red fluorescent protein orthotopic model of pancreatic cancer. Cancer Res. 63, 5521–5525 (2003).
Peyruchaud, O. et al. Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions. J. Bone Miner. Res. 16, 2027–2034 (2001).
Peyruchaud, O., Serre, C -M., NicAmhlaoibh, R., Fournier, P. & Clezardin, P. Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity. J. Biol. Chem. 278, 45826–45832 (2003).
Choy, G. et al. Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging. Biotechniques 35, 1022–1026 and 1028–1030 (2003).
Schmitt, C. A. et al. Dissecting p53 tumor suppressor functions in vivo. Cancer Cell 1, 289–298 (2002).
Schmitt, C. A. et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335–346 (2002).
Hasegawa, S. et al. In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis. Cancer Gene Ther. 7, 1336–1340 (2000). First paper to transform cancer cells in vivo with GFP. This is a model for future clinical applications of fluorescent proteins.
Kaneko, K. et al. Detection of peritoneal micrometastases of gastric carcinoma with green fluorescent protein and carcinoembryonic antigen promoter. Cancer Res. 61, 5570–5574 (2001).
Sato, Y. et al. In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate. FASEB J. 14, 2108–2118 (2000).
Varda-Bloom, N. et al. Tissue-specific gene therapy directed to tumor angiogenesis. Gene Ther. 8, 819–827 (2001).
Umeoka, T. et al. Visualization of intrathoracically disseminated solid tumors in mice with optical imaging by telomerase-specific amplification of a transferred green fluorescent protein gene. Cancer Res. 64, 6259–6265 (2004).
Qi, J., Link, C. J., Wang, S. Direct observation of GFP gene expression transduced with HSV-1/EBV amplicon vector in unfixed tumor tissue. Biotechniques 28, 206–208 (2000).
Chishima, T. et al. Visualization of the metastatic process by green fluorescent protein expression. Anticancer Res. 17, 2377–2384 (1997).
Lu, J -Y. et al. Establishment of red fluorescent protein-tagged HeLa tumor metastasis models: determination of DsRed2 insertion effects and comparison of metastatic patterns after subcutaneous, intraperitoneal, or intravenous injection. Clin. Exp. Metastasis 20, 121–133 (2003).
Wack, S. et al. Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo. Mol. Ther. 7, 765–773 (2003).
Weissleder, R., Tung, C. H., Mahmood, U. & Bogdanov, A. Jr. In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nature Biotechnol. 17, 375–378 (1999).
Bremer, C., Tung, C. H. & Weissleder, R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nature Med. 7, 743–748 (2001).
Jiang, T. et al. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc. Natl Acad. Sci. USA 101, 17867–17872 (2004).
Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004).
Verkhusha, V. & Lukyanov, K. A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nature Biotechnol. 22, 289–296 (2004). Review of new fluorescent proteins from the group that cloned the first RFP.
Zimmer, M. Green fluorescent protein (GFP): applications, structure and related photophysical behavior. Chem. Rev. 102, 759–781 (2002).
Yang, M., Luiken, G., Baranov, E. & Hoffman, R. M. Facile whole-body imaging of internal fluorescent tumors in mice with an LED flashlight. Biotechniques 39, 170–172 (2005).
Levenson, R., Yang, M. & Hoffman, R. M. Whole-body dual-color differential fluorescence imaging of tumor angiogenesis enhanced by spectral unmixing. Proc. Am. Assoc. Cancer Res. 45, 46 (2004).
Robert Hoffman is the President of AntiCancer Inc.
- CHRONIC-TRANSPARENT WINDOW
A tumour grown on the inner surface of the skin that can be viewed through a permanent opening in the animal, normally through a coverslip.
Literally 'correct surface'. The implantation of a tumour (or other tissue) into its organ of origin.
Passage of a cell from tissue into a blood or lymph vessel.
Movement of a cell out of the vasculature into interstitial spaces.
A molecule or part of a molecule that absorbs or emits light at specific wavelengths.
- EXTINCTION COEFFICIENT
The fractional absorbance of excitation light per unit path length of absorber.
- QUANTUM YIELD
The fraction of excited fluorophores that emit a fluorescence photon.
- INTRAVITAL VIDEO MICROSCOPY
In vivo microscopy of a live animal with images acquired in real time.
- MULTIPHOTON LASER-SCANNING MICROSCOPY
Multiphoton laser scanning microscopy (MPLSM) enables the production of long time-lapse recordings from live fluorescent specimens because it uses small beams of infrared light to illuminate only a small area of tissue at a time. So, in living tissue, damage is minimized and, because the light beam penetrates deeply, a greater volume of tissue can be examined.
- TRABECULAR BONE
Non-cortical spongy-bone-containing lacunae. Trabecular bone contains the bone marrow.
- TERMINAL PORTAL VENULES
Tributaries of the portal vein.
- CONFOCAL LASER-SCANNING MICROSCOPE
A microscope designed to minimize out-of-focus contributions from the vertical axis to an image. A pinhole aperture eliminates out-of-focus contributions.
- PRESINUSOIDAL VASCULATURE
Branches of the portal vein that leave the sinusoid.
- SEVERE COMBINED IMMUNODEFICIENT (SCID) MICE
Mice that are homozygous for the SCID mutation have compromised B-cell and T-cell immunity. This lack of immunity means that they can support human tumour xenografts for preclinical studies.
Apoptosis resulting from a lack of cellular adhesion.
- NUDE MICE
Strains of athymic mice bearing the recessive allele nu/nu that are mostly hairless and lack all, or most, of the T-cell population. Nude mice can accept either allografts or xenografts. nu/nu alleles on some backgrounds have near-normal numbers of T-cells.
Having the same genetic background, such as two cell lines that might differ only in a gene of interest.
- FLUORESCENCE STEREOMICROSCOPY
A microscope with two oculars that is equipped with a light source and filters for excitation of a fluorescence molecule and for the visualization of the resulting emission light.
- INTERNAL RIBOSOME ENTRY SITE
A region of DNA that codes for a messenger RNA sequence that can bind to ribosomes, which is often used to genetically link two proteins that would still be translated separately but be controlled by one promoter.
- NEAR INFRARED
The near-infrared region of the electromagnetic spectrum (covering a wavelength range of 700 nm to 3 nm) lies just beyond the sensitivity of the human eye.
About this article
Cite this article
Hoffman, R. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer 5, 796–806 (2005). https://doi.org/10.1038/nrc1717
Clinical & Experimental Metastasis (2020)
Beilstein Journal of Nanotechnology (2020)
Nano Research (2020)
Self-assembling as regular nanoparticles dramatically minimizes photobleaching of tumour-targeted GFP
Acta Biomaterialia (2020)
Scientific Reports (2020)