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  • 1. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 http://oncology.thelancet.com546 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. Lancet Oncol 2002; 3: 546–56 The study of microscopic cancer is essential for the understanding and control of cancer dormancy, growth, and colonisation of distant sites.1 Several approaches involving tumour-cell labelling have been developed for visualising tumour cells in vivo. The Escherichia coli beta-galactosidase (lacZ) gene has been used to detect micrometastases.2 However, lacZ detection requires extensive histological preparation and sacrifice of the tissue or animal. Therefore, other techniques are required for real-time imaging and study of tumour cells in viable fresh tissue or living animals. Use of skin-fold chambers, exteriorisation of organs, and subcutaneous windows inserted with semi-transparent material3–5 has yielded insights into microscopic tumour behaviour. However, these techniques are only suitable for ectopic models3 or for investigations with short periods of observation.4,5 The difficulties of maintaining windows or other devices that are made with heavy, stiff, or other types of foreign materials limit the length of time that these tools can be used in vivo.3,4 Cutaneous windows made with polyvinyl chloride film become opaque or detached after time, which precludes their use in long-term studies.4 However, in mice with green fluorescent protein (GFP)-expressing tumours (figure 1), gene expression, angiogenesis, and physiological properties can be studied by use of the dorsal skin-chamber combined with multiphoton confocal microscopy.3 A disadvantage to this technique is that the chamber limits investigation to the ectopic primary tumour. Mice bearing GFP-expressing microscopic tumours on exteriorised organs can be examined with intravital microscopy. However, with this approach, the mice tend not to survive long enough to enable spatial–temporal studies of tumour dormancy, progression, and metastasis.5 Weissleder and colleagues6,7 infused tumour-bearing animals with probes that fluoresce at an infrared frequency when activated by protease activity. Tumours with Review GFP imaging in vivo RMH is President of AntiCancer Inc and Professor of Surgery at the University of California, CA, USA. Correspondence: Dr Robert Hoffman, AntiCancer Inc, 7917 Ostrow Street, San Diego, CA 92111, USA. Tel: +1 858 654 2555. Fax: +1 858 268 4175. Email: Green fluorescent protein imaging of tumour growth, metastasis, and angiogenesis in mouse models Robert M Hoffman Figure 1. GFP transfectants in veins and capillaries. To study the limit of detection of GFP transfectants in vivo, a nude mouse was sacrificed 2 minutes after Chinese hamster ovary-GFP cells were injected into the tail vein. The fresh organ tissues removed from the mice were examined by use of fluorescence microscopy. GFP-expressing clone-38 cells formed emboli in a capillary of the right adrenal gland. Bar=100 ␮m.26
  • 2. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 547 appropriate proteases activated the probes and could be seen externally. This system is limited by very high liver-to-tumour background fluores- cence, which means that metastasis to the liver–among the most important metastatic sites–cannot be studied. Furthermore, the time limit for studies was 96 hours, so growth and efficacy studies were not possible. Tumours must have appropriate protease activity to be detectable, and the probes must be delivered selectively to the tumour. Another approach for visualising tumour cells in vivo involves insertion of the luciferase gene into tumour cells causing them to emit light.8,9 However, once transferred to mammalian cells, luciferase enzymes require exogenous delivery of their luciferin substrate, which is an impractical procedure in an intact animal. Because of the low image resolution and signal achieved with this approach, it takes a substantial amount of time to collect sufficient photons to form an image from an anesthetised animal.8 Also, whether luciferase genes can function stably in tumours over long periods and in the metastases derived from them, is not known. 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 requires no other Aequorea proteins, substrates, or cofactors to fluoresce.15 Recently, gain-of- function bright mutants expressing the GFP gene have been generated by various techniques16–19 and have been humanised for high expression and signal.20 Red fluorescence proteins (RFP) from the Discosoma coral have also been described and should prove useful for in vivo imaging studies.21–23 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).24–28 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 cancer,27,29–32 prostate cancer,33,34 melanoma,35 colon cancer,36 pancreatic cancer,37,38 breast cancer,39–43 ovarian cancer,26,44,45 and brain cancer.46,47 This review shows that tumour cells transfected with the GFP gene are a powerful tool for in vivo visualisation of tumour growth, angiogenesis, dormancy, dissemination, invasion, and metastasis. Ex vivo imaging of tumours in animal models 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 were detected on the lungs of all mice and multiple micrometastasis were also detected on the liver, kidney, contralateral ovary, adrenal gland, para-aortic lymph node, and pleural membrane. Single fluorescing cells could be visualised with GFP that could not be detected with standard histological techniques.26,44 Additional studies, in which CHO-K1-GFP cells were injected into the tail vein of nude mice, showed that tumours, and even single cells, could be detected by GFP fluorescence in the peritoneal wall vessels.26 These cells formed emboli in the capillaries of the lung, liver, kidney, spleen, ovary, adrenal gland, thyroid gland, and brain. Lung cancer GFP expressing cells from the highly metastatic human cell line ANIP were implanted by SOI onto the left lungs of nude mice.27,28 The advancing margin of the tumour, which spread through the ipsilateral lung, was visualised by GFP fluorescence. All the animals showed evidence of chest- wall invasion and local and regional spread. Metastatic contralateral tumours involved the mediastinum, contralateral pleural cavity, and the contralateral visceral pleura. Whereas the ipsilateral tumour had a continuous and advancing margin, the contralateral tumour seemed to have been formed by multiple seeding events. Involvement of contralateral hilar and cervical lymph nodes was also visualized by GFP expression. When non-GFP-transfected ANIP was compared with GFP-transformed ANIP for metastatic capability similar results were seen.27,28 Mice implanted by SOI with GFP-expressing human lung cancer cells (H-460-GFP) were sacrificed after 3–4 weeks ReviewGFP imaging in vivo Figure 2. Whole-body external images of murine melanoma (B16F0-GFP) metastasis in brain. The metastasis were imaged by GFP expression under fluorescence microscopy. Clear images of metastatic lesions in the brain can be seen through the scalp and skull. (a) 14 days after injection of GFP-expressing tumour cells. Bar=1280 ␮m. (b) 20 days after injection. Bar=1280 ␮m. (c) 25 days after injection. Bar=1280 ␮m.36
  • 3. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 http://oncology.thelancet.com548 when there was a significant decline in performance status. GFP fluorescence showed that there were metastases in the left lung, the contralateral lung, chest wall, and the skeletal system. The vertebrae were the most involved skeletal site of metastasis (figure 3); skull, tibia, and femur marrow metastasis were also observed by GFP fluorescence.29 Paris and co-workers30 described a model of spontaneous lung metastases of H-460M-GFP in nude mice. Nude mice were subcutaneously inoculated with H-460M-GFP cells and, after 2 weeks, 75% of animals had fluorescent spontaneous lung micrometastases. From the third week onwards, all the animals had metastatic disease, mainly in the lungs. By 7 weeks, the number of lung metastases had increased to about 400 in all mice. Chishima and co-workers injected human ANIP-GFP lung-cancer cells into the tail vein of nude mice, which were subsequently sacrificed after either 4 or 8 weeks.28 In both groups, numerous micrometastatic colonies in the lung were detected by GFP expression in fresh tissue.8 weeks after injection, most of the colonies were no more developed than those in mice sacrificed at 4 weeks.Several small colonies with 10 or fewer cells were detected at the surface of the lung in both groups. Dormant tumour cells were seen in the lung along with those that were actively colonising,28 which is an extremely interesting finding since micrometastatic dormancy is one of the most important processes for understanding tumour progression. After 8 weeks, metastases were present in the brain, the submandibular gland, lung, pancreas, bilateral adrenal glands, peritoneum, and pulumonary hilum lymph nodes.28 Lewis lung carcinoma cells have been used widely for many important studies, but many groups use subcutaneous transplant or orthotopic cell-suspension injection models, which do not allow the tumour to fulfill its metastatic potential. However, in studies involving a new highly metastatic model of Lewis lung carcinoma, which has been developed by use of SOI and GFP-expressing tumours, 100% of animals had metastases on the ipsilateral diafragmatic surface, contralateral diafragmatic surface, contralateral lung, and in mediastinal lymph nodes. Heart metastases were seen in 40% of the animals, and brain metastases were present in 30%.31 Hastings and co-workers investigated the effects of treatment with a neutralising antibody to parathyroid related hormone (PTHrP) on the growth of BEN cells—a human lung squamous-cell carcinoma line that expresses PTHrP and its receptor. BEN-GFP cells were implanted into athymic mice producing orthotopic lung tumours. The mice received either subcutaneous PTHrP antibodies or irrelevant immunoglobulin. After 30 days, six of 10 mice receiving antibodies to PTHrP had GFP-expressing lung tumours, whereas only one of the 10 control mice had developed cancer.32 Prostate cancer GFP-labelled human prostate cancer cells (PC-3-GFP) were implanted by SOI in the dorsolateral lobe of the prostate in nude mice.33 GFP fluores- cence indicated the presence of skeletal metastasis in several places including the skull, rib, pelvis, femur, and tibia. The tumours also metastasised to the lung, pleural membrane, kidney, liver, adrenal gland, brain, and spinal cord.33 Severe combined immunodeficient (SCID) mice orthotopically inoculated Review GFP imaging in vivo Figure 3. External image of bone metastasis of AC3488-GFP. External images of tumours in the skeletal system including (a) the skull, (b) scapula, and (c) spine in a dorsal view of live intact nude mouse.36 Figure 4. External imaging of single U-87 glioma cells in microscopic tumour colonies on the brain imaged through a skin flap in a nude mouse. Single cancer cells and small colonies of cancer cells can be seen. Bar=80 ␮m.54
  • 4. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 549 with LNCaP-GFP cells and metastases to distant organs were chronologically examined.34 LNCaP-GFP cells were detected in the lung under fluorescence microscopy as early as 4 weeks after inoculation. There was no difference in growth rates and androgen-responsiveness between LNCaP-GFP and LNCaP cells observed in vitro. LNCaP-GFP cells inoculated in SCID mice produced prostate-specific antigen. Melanoma Malignant melanoma cell lines that highly express GFP (B16-GFP) were used to visualise skeletal and multiorgan metastases in two types of mouse. C57BL/6 mice received an intravenous injection of B16-GFP cells and nude mice were given an intradermal injection of human melanoma LOX.35 The animals were sacrificed after 3 weeks and extensive B16F0 metastases to the bone and bone marrow were visualised by use of GFP expression. For both cell lines, metastases were present in several organs including brain, lung, pleural membrane, liver, kidney, adrenal gland, lymph nodes (figure 7), skeleton, muscle, and skin. Moore and colleagues50 also implanted B16-GFP into the hind leg of mice; all animals developed para-aortic lymph node metastases. Pancreatic cancer GFP-expressing metastases of two human pancreatic cancer cell lines, MIA-PaCa2-GFP and BxPC-3-GFP, were examined in orthotopic models. The cells were transplanted by SOI to the pancreas of nude mice and GFP expression was used to visualise the chronology of pancreatic tumour growth and metastasis. BxPC-3-GFP tumours developed rapidly and spread to the spleen and retroperitoneum as early as 6 weeks after inoculation. However, distant metastases were rare. By contrast, MIA- PaCa-2-GFP tumours grew slowly in the pancreas, but rapidly metastasised to distant sites including the liver and portal lymph nodes. Regional metastases with these tumours were rare. These studies showed that pancreatic cancers have highly specific and individual “seed–soil” interactions, which governs the chronology and sites of metastases.37 Brain cancer Daoy medulloblastoma-GFP cells were stereotactically injected into the frontal cortex of nude mice by MacDonald and colleagues.46 The primary tumour margins were clearly visible in brain tissue sections by GFP fluorescence; local invasion and micrometastases, including single cells, could also be visualised. However, the micrometastases could not be detected by routine hematoxylin and eosin staining and immunohistochemistry. Breast cancer Schmidt and colleagues implanted GFP-expressing human breast tumour cells (MDA-MB-435-GFP) orthotopically in the mammary fat pad of SCID mice.39 Metastatic cells migrated to the lungs of the animals very early after orthotopic implantation and the numbers of migrating cells continued to increase until death, about 8 weeks later. By ReviewGFP imaging in vivo Figure 5. External imaging of angiogenesis of orthotopic BxPC-3 human pancreatic tumour through a skin flap. The blood vessels can be seen by contrast with the GFP-expressing tumour.54 (a) Macroimaging of BxPC-3- GFP human pancreatic tumour angiogenesis through the abdominal wall skin flap window. (b) Microscopic imaging of BxPC-3-GFP angiogenesis through the abdominal-wall skin-flap window (magnification X20). (c) Microscopic imaging of Bx PC-3-GFP angiogenesis through the abdominal-wall skin-flap window (magnification X40).
  • 5. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 http://oncology.thelancet.com550 use of flow cytometry, one GFP-fluorescent tumour cell per 200 000 host cells could be detected in dissociated tissues. Circulating tumour cells in blood could also be visualised by GFP fairly soon after implantation.39 Human MDA-MB-435-GFP breast cancer cells, which are tumorigenic but slow-growing in vivo, were transfected with extracellular matrix metalloproteinase inducer cDNA. The transfected cells were injected orthotopically into mammary tissue of female NCr nude mice41 and tumour growth was compared with empty plasmid-transfected cancer cells. GFP fluorescence confirmed that the cDNA- transfected cells were considerably more tumorigenic and invasive than the plasmid-infected cells. GFP-expressing cells have also been used to visualise tumour-cell motility in orthotopic models of breast tumours in rats.40 A metastatic rat breast-cancer cell line was established that constitutively express- ed GFP. These cells were injected into the mammary fat pad of female Fischer 344 rats and, after skin had been removed from the animals, primary and metastatic tumours were visualised by GFP fluorescence. By use of a laser scanning confocal micro- scope, intravital imaging of the primary tumour in situ enabled the motility patterns of the tumour cells to be visualised.40 Cortactin is a substrate of the Src-related tyrosine kinases, which is known to enhance tumour progression in MDA-MB-231 breast cancer cells. When nude mice were injected through their cardiac ventricles with MDA-MB-231 cells overexpressing GFP-cortactin, the animals showed bone osteolysis at a rate that was about 85% higher than in animals injected with cells expressing the vector alone.43 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 preferentially grow and survive near the liver surface. A small population of single cells persisted over the 11-day observation period and the investigators believe these cells may represent dormant tumour cells.5 Rat tongue carcinoma cell lines expressing GFP have been used to investigate the formation of micrometastasis. The cells were injected into the portal vein and then tracked by use of intravital video microscopy.51 The two cell types— LM-GFP metastatic and E2-GFP non-metastatic tongue carcinoma cells—immediately got stuck in the sinusoidal vessels near terminal portal venules. The E2-GFP cells disappeared from the liver sinusoid within 3 days, whereas a substantial number of LM-GFP cells remained in the liver— possibly because these cells formed stable attachments to the sinusoidal wall. Upon examination of the process with a confocal laser scanning microscope, only LM-GFP cells were shown to grow in the liver. Review GFP imaging in vivo Figure 6. Whole-body fluorescence imaging of lymphoma dissemination. A mouse lymphoma arising in a Em-myc transgenic mouse was transduced with GFP and introduced to syngenic mice.71 (a) Lymphoma disseminated to lymph nodes, bone, and brain can be seen. (b) The rendered figure eliminates all non-GFP-expressing areas of the mouse with the z-direction indicating intensity.
  • 6. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 551 GFP techniques have also shown that CT-26 (mouse colon carcinoma) cells inhibit the development of liver metastasis in BALB/c mice that receive intraportal injections of GFP-expressing tumour cells.52 Intravital microscopy of the livers of these mice showed that growth of primary tumours promoted dormancy of single tumour cells for up to 7 days. Immunohistological staining for Ki-67 confirmed that these solitary cells were indeed dormant. By contrast, in the absence of a primary tumour, GFP-expressing tumour cells quickly developed into micrometastases. Thus, primary CT-26 tumour implants seem to inhibit tumour metastasis by promotion of a state of single-cell dormancy. The ras oncogene promotes growth of micrometastases into macroscopic metastases. Two types of cell with constituitively active ras, NIH 3T3 and T24 H-ras- transformed (PAP2) fibroblasts, both of which were expressing GFP, were injected into mouse liver. Subsequent examination by GFP intravital imaging established that only the micrometastases formed by ras-transformed cells went on to produce macroscopic metastases; most NIH 3T3 micrometastases just disappeared. Furthermore, PAP2 met- astases had a significantly higher proportion of proliferating cells than apoptotic cells, whereas NIH 3T3 metastases had low proliferation and a high proportion of apoptotic cells.53 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 fluorescent. This can be accomplished by in vitro26,36 and in vivo64 selection of such fluorescent tumour cells. To produce metastasis in mice, the genetically-fluorescent tumours should be transplanted orthotopically.26,27,29,31–34,37,38,44,48 Once the GFP-expressing tumours have developed and metastases have formed, individual tumour cells can be detected in the live mouse by use of whole-body imaging with fairly simple equipment. A fluorescence light box with fibre-optic lighting at about 490 nm and appropriate filters, placed on top of the light box, can be used to image large tumours and can be viewed with the naked eye.36 Alternatively, the light box can be linked to a camera with an appropriate filter to enable images to be displayed on a monitor and digitally stored.36 In order to visualise smaller tumours and metastases, the animal can be put on a fluorescence dissecting microscope which incorporates a light source and filters for excitation at about 490 nm. Fluorescence emission can be observed through a 520 nm long-pass filter.36 The animals can be irradiated at 490 nm for long periods without harming them or bleaching the GFP or RFP fluorescence. Images can be processed with standard software and the imaging procedures can be repeated as often as necessary without harming the animal. Therefore, with these techniques real- time tracking of tumour growth and metastasis is feasible. Reversible skin-flaps can also be introduced onto different parts of the animal in order to look at single tumour cells or small colonies on internal organs.54 The skin-flaps are rendered reversible by simple suturing. External imaging can then be done through the relatively transparent body walls of the mouse, which include the skull, by use of a fluorescence dissecting microscope. Blood vessels growing on tumours can also be observed using skin flaps because they contrast with the fluorescence of the tumours.54 Examples of findings from studies of these techniques are described below. 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 GFP-expressing tumours and their metastases can be obtained in freely- moving animals.29 In another study, GFP-expressing human pancreatic tumour cell lines were introduced as tissue fragments into the pancreases of nude mice by SOI. As the tumours were growing, the investigators used whole-body optical imaging techniques to track, in real-time, the growth of the primary tumour and the formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. The images were used for quantification of tumour growth in each of these organs.38 Using a different approach, human ovarian tumour cells (SKOV3.ip1) were made to express GFP by infection with a replication-deficient adenoviral (Ad) vector encoding GFP.45 The infected cells showed high GFP fluorescence, and when implanted into mice, intraperitoneal tumours as small as 0.2 mm in diameter could be detected externally. Peyruchaud and colleagues established a GFP-expressing bone-metastasis subclone of MDA-MB-231 (B02/GFP.2) by repeated in vivo passages in bone, by use of the heart injection model. When injected into the tail vein of mice, the selected cells grew preferentially in bone. Whole-body fluorescence imaging of the live mice showed that bone metastases could be detected about 1 week before radiologically distinctive osteolytic lesions developed. Furthermore, when the tumour- bearing mice were treated with a bisphosphonate, progression of established osteolytic lesions, and the expansion of breast cancer cells within bone, were inhibited.42 Opening a reversible skin-flap in the light path greatly reduces attenuation of the fluorescent signal, thereby increasing the sensitivity of tumour detection by many times. This procedure also greatly increases the depth at which tumour cells can be observed. Single GFP-expressing tumour cells can thus be seen on numerous internal organs. GFP glioma cells seeded on the brain can be visualised through a scalp skin-flap (figure 4). GFP lung tumour micro-foci, which represent a few malignant cells, can be ReviewGFP imaging in vivo
  • 7. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 http://oncology.thelancet.com552 viewed through a skin-flap over the chest wall and contralateral micrometastases can also be examined by use of the corresponding skin-flap. Peritoneal wall skin flaps can be used to look at GFP-pancreatic tumours and their angiogenic micro-vessels (figure 5). For the liver, a skin-flap allowed imaging of physiologically relevant micro- metastases that had originated in an orthotopically- implanted GFP tumour. Single tumour cells on the liver, which had been introduced through an intraportal injection, were also detectable.54 Ilyin and co-workers visualised glioma cells in rats by inserting a fibre-optic endoscope through a pre-implanted guide cannula. Tumour monitoring was coupled to confocal microscopy so that visualisation of the fluorescent signals from the C-6 glioma-GFP cells that had been preimplanted in the brain, was very sensitive.47 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 that capillary density increased linearly over 10 days. Similarly, when GFP-expressing MDA-MB-435 cells were ortho- topically transplanted to the mouse fat pad, whole-body optical imaging showed that blood vessel density increased linearly over about 20 weeks. As described above, reversible skin flaps can be used to visualise angiogenesis on GFP-expressing tumours transplanted onto internal organs (figure 5), in addition to examining the tumour itself.54 These angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumour angiogenesis.48,54 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 orthotopic mammary-pad injection model, Wyckoff and colleagues65 also visualised tumour–vessel interaction by looking at the fluorescence of the tumour cells. Al-Mehdi1 and co-workers saw what they claimed to be intravascular tumour colony formation by tracking GFP expression in a lung perfusion study. In addition, Moore and colleauges have also visualised vessels in a GFP-expressing rodent cell line.49 Wong and colleagues57 showed that death of trans- formed, metastatic, rat embryo cells, which were expressing GFP, occurred via apoptosis in the lungs 24–48 h after in- jection into the circulation. The researchers established that BCL-2 overexpression was causing inhibition of apoptosis in culture, and this mechanism also conferred resistance in vivo for 24–48 h after injection. This inhibition of apoptosis led to a greater number of macroscopic metastases. Large detectable metastases did not form after athymic mice were given an intravenous injection of chromosome 6- transduced tumour cells expressing GFP.58 However, fluorescence microscopy revealed micrometastases (single cells or clusters of fewer than 10 cells) in the lungs, suggesting that these cells managed to lodge themselves in the lungs, but failed to proliferate. Cells isolated from mouse lungs up to 60 days after the injection were able to grow in culture and formed tumours when injected into skin; therefore, the cells were still viable, but dormant. This result implies that the gene(s) on chromosome 6 interfere specifically with growth regulatory response in the lung, but not in the skin. Chang and co-workers59 used CD31 and CD105 to identify endothelial cells and GFP-labelling of tumour cells and showed that colon carcinoma xenografts had mosaic vessels with focal regions where no CD31/CD105 immunoreactivity was detected and tumour cells appeared to contact the vessel lumen. 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 microscopy showed that the tumour was able to induce activity of the VEGF promoter; GFP-positive stromal cells were seen at least 200 ␮m into the tumour with this technique. Fukumura’s group also developed a novel in vivo microscopy technique to simultaneously measure VEGF promoter activity, pO2, and pH.62 To monitor VEGF expression in vivo, the investigators engineered human glioma cells that expressed GFP under the control of the VEGF promoter. The cells were subsequently implanted into cranial windows in SCID mice and VEGF promoter activity was assessed by GFP imaging. Findings from these studies suggest that VEGF transcription in brain tumours is regulated by both tissue pO2 and pH via distinct pathways. 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 experi- mental mouse tumours was undetectable. However, the tumour cells started to fluoresce brightly in response to an Review GFP imaging in vivo
  • 8. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 553 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 tumour growth and metastasis formation could be visualised by GFP expression. No normal tissues were transduced by the GFP retrovirus. 2 weeks after retroviral GFP delivery, GFP-expressing tumour cells were observed in the gonadal fat, greater omentum, and intestine, indicating that the tumours were efficiently transduced by the GFP gene and could be detected by its expression. Second and third laparotomies were done at weeks 4 and 6, respectively. GFP- expressing tumour cells were found spreading to lymph nodes in the mesentery. The fourth laparotomy, at 8 weeks, showed widespread tumour growth including metastasis to the liver. Thus, reporter-gene transduction of the primary tumour enabled detection of its subsequent metastasis. This reporter-gene therapy model could be applied to primary tumours before resection or other treatment and thus provide an early detection system for metastasis and recurrence.64 To determine whether the carcino-embryonic antigen promoter could direct the GFP gene, human MKN45-GFP stomach-cancer cells were injected into the peritoneal cavity of BALB/c nude mice.65 A CEA-EGFP plasmid was then introduced in the peritoneal cavity using liposomes. GFP- fluorescent tumour nodules were subsequently detected by fluorescence stereomicroscopy. In another approach, GFP was conjugated to transferrin in order to target disseminated tumours in vivo.66 When GFP-gene conjugates were systemically administered through the tail vein to nude mice that had been subcutaneously inoculated with tumour, GFP expression was only detected only in the tumour. Varda-Bloom and colleagues67 developed a tissue- specific gene therapy to the angiogenic blood vessels of tumour metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter driving GFP. High and specific activity of PPE-1was achieved by systemic administration of the adenoviral vector to mice bearing Lewis lung carcinoma tumours. This effect was detected by GFP expression in the new vasculature of primary tumours and lung metastasis. The highest area of expression was in the angiogenic endothelial cells of the metastasis. Recently, GFP on an HSV-1/EBV vector has been administered to tumour-bearing animals. However, persistant GFP expression was not achieved in this study.68 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 occurred at the stem-cell level. Such an approach could be used clinically in the future to protect bone marrow against chemo- therapy.70 p53 tumour-suppressor functions were visualised in vivo by GFP imaging (figure 6). By use of the antiapoptotic gene BCL-2 or a dominant-negative caspase 9 (C9DN), GFP whole-body imaging established that disruption of the apoptosis pathway downstream of p53 leads to Eµ-myc-GFP lymphoma expansion that phenocopies p53 loss in Eµ-myc transgenic mice. This finding shows that GFP whole-body imaging can be used to identify individual genes that affect tumour growth. Such information could be used to identify genes predicting aggressive tumour growth.71 A senescence programme controlled by p53 and p16INK4a 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 p16INK4a . 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 tumours with senescence defects. These findings indicated that premature senescence can contribute to treatment outcome in vivo and provide new insights into the molecular genetics of drug resistance, which can be applied clinically.72 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 ReviewGFP imaging in vivo
  • 9. For personal use. Only reproduce with permission from The Lancet Publishing Group. THE LANCET Oncology Vol 3 September 2002 http://oncology.thelancet.com554 cells studied so far and is exemplified by the generatation of extensive GFP-expressing metastases. The use of GFP-expressing tumour cells in transplanted mice, fresh tissue, or live animals5,26 has provided new insights into the real-time growth and metastatic behaviour of cancer. Several independent studies, which include an extensive comparison between metas- tases of GFP-transduced rat mammary carcinoma cell and the parental cell line,26,28,40 have shown that GFP transduction and expression does not affect metastatic behaviour.40 GFP can be transfected into any cell type of interest and used as a cytoplasmic marker to show the general outlines of cells in vivo and fine morphological details such as long slender pseudopodial projections.5 The development of tumour cells that stably express GFP at high levels has enabled investigation of tumour and metastastic growth in a completely non-invasive manner, by use of whole-body imaging.36 For the first time, tumour growth and metastatic studies, including drug evaluations, can be done and quantified in real-time in individual animals—the potential of this technology is very high. A further advantage of GFP-expressing cells is the increased contrast between brightly fluorescent tumour tissue and blood vessels within it. The ability to visualise and quantify blood-vessel development in metastases in vivo will greatly facilitate studies of angiogenesis and the testing of effects of antiangiogenic agents on metastatic deve- lopment.5,48 The GFP approach has several important advantages over other optical approaches to imaging (table 1). In comparison with the luciferase reporter, GFP has a much stronger signal and therefore can be used to image unrestrained animals. The fluorescence intensity of GFP is very strong since the quantum yield is about 0·9.15–19 The protein sequence of GFP has also been “humanised”, which enables it to be highly expressed in mammalian cells.20 In addition, GFP fluorescence is fairly unaffected by the external environment since the chromaphore is protected by the three-dimensional structure of the protein.13 The excitation wavelength is quite long at 490 nm,16–19 which does not quench the fluorescence and therefore long-term measurements can be made. In vivo, GFP fluorescence is mainly limited by light scattering which, as noted above, can be overcome by skin flaps54 and endoscopes47 such that single cells can be imaged externally. Longer wavelength fluorescence proteins, such as RFP, can also be used to reduce scatter. The luciferase reporter technique, however, requires that animals are anesthetised and restrained so that sufficient photons to construct an image can be collected. Furthermore, this process must be carried out in a virtually light-free environment and animals must be injected with the luciferin substrate, which has to reach every tumour cell in order to be useful. This limitation precludes studies that would be perturbed by anesthesia, restraint, or substrate injection and also makes high-throughput screening unfeasible. Expression of firefly luciferase (Luc) can be used to visualise tumour-cell growth and regression in response to various therapies in mice. However, detection of Luc-labelled cells in vivo was limited to 103 human tumour cells.8,9 The high intensity signal produced by GFP, however, allows unrestrained animals to be imaged without any perturbation or substrate—irradiation with non-damaging blue light is the only step needed. Images can be captured with fairly simple apparatus and there is no need for darkness. Near-infrared probes activated by the action of proteases6,7 can also be used for optical imaging of tumours. This approach requires substrate injection and the tumour must contain a specific protease that cleaves the substrate. Tumours on normal tissues, eg, the liver, that contain these proteases can not be visualised because background signals are too high, so that there is not sufficient distinction between tumour and normal tissue to obtain useful external images.73 For clinical application of GFP developments, future studies may make use of the approach of Hasegawa and colleagues,64 who used retroviral GFP vectors that were selectively targeted to tumours for the purpose of identifying future metastasis. GFP labelling of tumour-infiltrating lymphocytes69 and bone marrow transduced with GFP- linked genes that confer chemoresistance,70 also have intriguing clinical potential. Conflict of interest RMH is President of AntiCancer Inc—the company that commercially exploits two mouse models (the MetaMouse and the AngioMouse) for visualisation of tumour spread and angiogenesis by use of GFP- expression. Many of the studies discussed in this review make use of this technology. References 1 Al-Mehdi AB, Tozawa K, Fisher AB, et al. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat Med 2000; 6: 100–02. 2 Lin WC, Pretlow TP, Pretlow TG 2nd , Culp LA. Bacterial lacZ gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. Cancer Res 1990; 50: 2808–17. Review GFP imaging in vivo 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. Table 1. Comparison of opitical imaging methods for tumour growth and metastasis Method Minimum Minimum Need for Need for Method of Multi- cells cells substrate? anesthesia? visualisation colour imageable imageable imaging in vitro in vivo GFP 1* 1*† No† No§ Direct Imaging‡ Yes† Luciferase 300|| 3000¶ Yes¶ Yes¶ Photon counting (pseudo-colour) No *Reference 26; †reference 54; ‡reference 36; §reference 74; ||reference 75; ¶reference 76.
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