The European Union recently awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease

Francis Hamilton, the Briton who first described zebrafish ( Danio rerio ) in 1822, would be astounded to see the scientific attention now afforded to this two-inch-long native of Indian rivers. A fish with no economic worth was how he described this little creature. Yet recently, the European Union awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease. When and why did zebrafish swim from home aquaria into research labs, and what can we learn about our biology from this surprising source?The Early Days It was the late 1960s when phage geneticist George Streisinger began to look for a model system in which to study the genetic basis of vertebrate neural development. His passion for tropical fish led him to the humble zebrafish. He was a ‘visionary’, remembers neurobiologist Judith Eisen (University of Oregon, Eugene, Oregon, United States), ‘who laid the groundwork for the use of zebrafish as a developmental model’. Eisen, who now heads her own research group, went to Oregon in 1983 to work on  Xenopus  neural development but soon became attracted to zebrafish as a model organism. By the early 1980s, she explains, Streisinger had worked out many of the genetic tricks needed to tackle zebrafish development. What's more, the fish had ‘wonderful embryology’. The embryo, which develops outside its mother, is transparent. ‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists.
The Early Days
It was the late 1960s when phage geneticist George Streisinger began to look for a model system in which to study the genetic basis of vertebrate neural development. His passion for tropical fish led him to the humble zebrafish. He was a ‘visionary’, remembers neurobiologist Judith Eisen (University of Oregon, Eugene, Oregon, United States), ‘who laid the groundwork for the use of zebrafish as a developmental model’.Eisen, who now heads her own research group, went to Oregon in 1983 to work on  Xenopus  neural development but soon became attracted to zebrafish as a model organism. By the early 1980s, she explains, Streisinger had worked out many of the genetic tricks needed to tackle zebrafish development. What's more, the fish had ‘wonderful embryology’. The embryo, which develops outside its mother, is transparent. ‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists.Fast Forward to the Big Screen The properties of zebrafish that attracted Eisen soon attracted people interested in other aspects of vertebrate development to the stripy tiddler ( Figure 1 ). As Eisen comments: ‘No other developmental model has risen to prominence so quickly’. These days more than 3,000 researchers are listed on ZFIN, a United States–based information resource for the zebrafish research community. Figure 1 Adult Zebrafish (Image courtesy of Lukas Roth, University College London, London, United Kingdom.) The speedy expansion was driven in great part by two genetic screens initiated in 1992–1993 by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever and Mark Fishman in Boston, Massachusetts. The aim of both screens was to identify genes with unique and essential functions in zebrafish development, and in 1996 an issue of the journal  Development  was dedicated to the mutants that had been isolated and characterised. These screens, says Ralf Dahm (Max Planck Institute for Developmental Biology, Tübingen, Germany), project manager of the ZF-MODELS consortium, ‘were the first major zebrafish projects, and they showed that zebrafish was a model organism to be reckoned with’. ‘That was a fantastic time’, says Derek Stemple, then a postdoc with Driever but now a group leader at the Wellcome Trust Sanger Institute (Cambridge, United Kingdom) and a ZF-MODELS participant. ‘From Wolfgang's lab, I was able to take the mutations that affected notochord development, and have been studying them ever since’. The notochord is an embryonic structure that forms the primitive axial skeleton of the developing embryo, and because mutations affecting notochord development result in shortened embryos, seven of the affected genes have been named after the dwarves in Snow White—zebrafish, like some other developmental models, have many imaginatively named mutants. Stemple now knows the identity of six of these mutated genes, all of which lead to disruption of basement membrane around the notochord. Many mutants from those first two screens are still used by developmental biologists, but another set of mutants has recently been isolated by Nancy Hopkins, Amgen Professor of Biology at the Massachusetts Institute of Technology (Cambridge, Massachusetts, United States). About ten years ago, Hopkins started to develop insertional mutagenesis in zebrafish ( Figure 2 ). In this approach, mutations are caused by the random insertion of viral DNA throughout the fish genome. The inserted DNA acts as a tag, making cloning of mutated genes very straightforward, although the efficiency of the initial insertional mutagenesis is much lower than that of the chemical mutagenesis used in the 1992–1993 screens. Hopkins has isolated 550 mutants in her screen, representing around 400 different genes, and has cloned more than 300 of these genes to date. Some of the fruits of this project are published in this issue of PLoS Biology. Hopkins's group is now collaborating with 25 external laboratories on the annotation of the mutant collection with funding from the National Center for Research Resources, part of the United States National Institutes of Health. Figure 2 A Zebrafish Pigment Mutant The mutant called bleached blond was produced by insertional mutagenesis. The embryos in the picture are four days old. At the top is a wild-type embryo, below is the mutant. The mutant lacks black pigment in the melanocytes because it fails to synthesise melanin properly. (Image courtesy of Adam Amsterdam, Massachusetts Institute of Technology, Boston, Massachusetts, United States.) The Tübingen researchers did another chemical mutagenesis screen between 2000 and 2001, and are now starting a third screen of 6,000 genomes as part of the ZF-MODELS project. ‘Each of our screens has built on the previous one by including more specific assays’, explains Dahm. Mutagenesis for the third screen is underway, but the assays, which include looking for defects that specifically affect adults, are still at the pilot stage; this autumn, the project's executive committee, which is headed by Nüsslein-Volhard, will decide which assays to use in the full-scale screen. ‘Just over half the 17 partners in the consortium will come to Tübingen to do screens’, predicts Dahm. ‘By bringing in expertise in different systems in this way we should greatly increase the efficiency of the screen’.
Fast Forward to the Big Screen
The properties of zebrafish that attracted Eisen soon attracted people interested in other aspects of vertebrate development to the stripy tiddler ( Figure 1 ). As Eisen comments: ‘No other developmental model has risen to prominence so quickly’. These days more than 3,000 researchers are listed on ZFIN, a United States–based information resource for the zebrafish research community.The speedy expansion was driven in great part by two genetic screens initiated in 1992–1993 by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever and Mark Fishman in Boston, Massachusetts. The aim of both screens was to identify genes with unique and essential functions in zebrafish development, and in 1996 an issue of the journal  Development  was dedicated to the mutants that had been isolated and characterised. These screens, says Ralf Dahm (Max Planck Institute for Developmental Biology, Tübingen, Germany), project manager of the ZF-MODELS consortium, ‘were the first major zebrafish projects, and they showed that zebrafish was a model organism to be reckoned with’.Development‘That was a fantastic time’, says Derek Stemple, then a postdoc with Driever but now a group leader at the Wellcome Trust Sanger Institute (Cambridge, United Kingdom) and a ZF-MODELS participant. ‘From Wolfgang's lab, I was able to take the mutations that affected notochord development, and have been studying them ever since’. The notochord is an embryonic structure that forms the primitive axial skeleton of the developing embryo, and because mutations affecting notochord development result in shortened embryos, seven of the affected genes have been named after the dwarves in Snow White—zebrafish, like some other developmental models, have many imaginatively named mutants. Stemple now knows the identity of six of these mutated genes, all of which lead to disruption of basement membrane around the notochord.Many mutants from those first two screens are still used by developmental biologists, but another set of mutants has recently been isolated by Nancy Hopkins, Amgen Professor of Biology at the Massachusetts Institute of Technology (Cambridge, Massachusetts, United States). About ten years ago, Hopkins started to develop insertional mutagenesis in zebrafish ( Figure 2 ). In this approach, mutations are caused by the random insertion of viral DNA throughout the fish genome. The inserted DNA acts as a tag, making cloning of mutated genes very straightforward, although the efficiency of the initial insertional mutagenesis is much lower than that of the chemical mutagenesis used in the 1992–1993 screens. Hopkins has isolated 550 mutants in her screen, representing around 400 different genes, and has cloned more than 300 of these genes to date. Some of the fruits of this project are published in this issue of PLoS Biology. Hopkins's group is now collaborating with 25 external laboratories on the annotation of the mutant collection with funding from the National Center for Research Resources, part of the United States National Institutes of Health.The Tübingen researchers did another chemical mutagenesis screen between 2000 and 2001, and are now starting a third screen of 6,000 genomes as part of the ZF-MODELS project. ‘Each of our screens has built on the previous one by including more specific assays’, explains Dahm. Mutagenesis for the third screen is underway, but the assays, which include looking for defects that specifically affect adults, are still at the pilot stage; this autumn, the project's executive committee, which is headed by Nüsslein-Volhard, will decide which assays to use in the full-scale screen. ‘Just over half the 17 partners in the consortium will come to Tübingen to do screens’, predicts Dahm. ‘By bringing in expertise in different systems in this way we should greatly increase the efficiency of the screen’.What Else Will ZF-MODELS Do? The ZF-MODELS consortium, which is funded under the European Union's Sixth Framework Programme, aims to establish zebrafish models for human diseases, discover genes that will lead to the identification of new drug targets, and gain fundamental insights into human development. ‘We will mainly focus on using advanced technologies that have recently become available’, says scientific coordinator Robert Geisler (Max Planck Institute for Developmental Biology). For example, Geisler's lab will use DNA chip technology to investigate gene expression patterns in zebrafish mutants and so provide increased knowledge of the regulatory pathways that act in zebrafish development. Consortium members will also use ‘reverse genetics’ to investigate these pathways. In reverse genetics, researchers start with a gene of interest and investigate the phenotypic effect of altering its activity; by contrast, in ‘forward genetics’ the starting point is to look for a particular phenotype and then hunt out the altered gene that is causing it. Two reverse genetics approaches will be used by the consortium. Gene expression will be transiently knocked down with morpholinos, short segments of the gene that block its function. In addition, a recently developed technique known as TILLING (targeting induced local lesions in genomes) will be used to knock out gene activity permanently. The first step in TILLING is to mutagenise male zebrafish and mate them with untreated female fish, explains Stemple, whose group is one of three ZF-MODELS partners who will use this approach. Offspring are raised to adulthood, and the DNA of each individual is then genotyped for the exon of interest. The consortium already has a collection of 6,000 such individuals, and once a fish carrying a mutation in the gene of interest has been identified, it will be outcrossed to produce offspring, half of which will carry the desired mutation on one of their chromosomes. ‘It is then a matter of identifying these heterozygote fish and incrossing them to get homozygous fish in which you can see the phenotype that correlates with that mutation’, says Stemple. As well as helping to produce knock-outs for other researchers, Stemple is also using the TILLING technique to develop zebrafish models for muscular dystrophy. Among the genes that are important in notochord development are those that encode laminins. This led Stemple into studying muscular dystrophy because laminins are involved in the human disease. ‘When we used morpholinos to disrupt [the production of] dystroglycan, a laminin receptor, we got a good model for muscular dystrophy’, he explains. Now, he plans to use TILLING to disrupt up to 30 other genes known to be involved in human muscular dystrophy. ‘In particular, we will look for hypomorphic mutants, fish that are viable but on the edge of falling apart’. These mutants can be used to identify small molecules that push the fish into muscular dystrophy. Finding molecules that can cause a disease in this way ‘might give us a handle on something to fix the disease’, says Stemple. In another strand of the ZF-MODELS project, zebrafish expressing green fluorescent protein (GFP) in specific cells or tissues will be generated and characterised ( Figure 3 ). In such fish, developing structures can be easily imaged over time in the living embryo. One researcher working on this aspect of the project is Stephen Wilson, Professor of Developmental Genetics at University College London (United Kingdom). GFP lines can be made either by attaching to the GFP gene regions of DNA that control, or ‘drive’, GFP expression in selected cell types or by allowing the GFP gene to insert randomly in the genome and looking for fish with specific expression patterns. ‘There are now many lines of fish available with different GFP expression patterns’, says Wilson, ‘and it is important to catalogue their expression so that people can use the most appropriate lines for their research’. Figure 3 Zebrafish Hindbrain (Left) Dorsal view of GFP-expressing neurons in the hindbrain of a one-day-old zebrafish embryo. (Right) Antibody-labelled axons. (Image courtesy of Dave Lyons, University College London.) Wilson's own interest is in neuroanatomy. Together with Jon Clarke, another developmental neurobiology group leader at University College London, he plans to analyse GFP lines in which small groups of neurons or particular parts of neurons are labelled, and in this way start to build a detailed reconstruction of early brain neuroanatomy. This, in combination with other work on zebrafish carrying mutations affecting neural development, will give the team ‘a better picture of how a vertebrate brain is built’. A final, important aspect of the ZF-MODELS project, adds Dahm, is database construction. ‘We will be developing a set of databases that will integrate all of the project data’, he explains. ‘In addition, we hope to integrate our data with that of ZFIN in the United States to make one central zebrafish resource’.
What Else Will ZF-MODELS Do?
The ZF-MODELS consortium, which is funded under the European Union's Sixth Framework Programme, aims to establish zebrafish models for human diseases, discover genes that will lead to the identification of new drug targets, and gain fundamental insights into human development. ‘We will mainly focus on using advanced technologies that have recently become available’, says scientific coordinator Robert Geisler (Max Planck Institute for Developmental Biology). For example, Geisler's lab will use DNA chip technology to investigate gene expression patterns in zebrafish mutants and so provide increased knowledge of the regulatory pathways that act in zebrafish development.Consortium members will also use ‘reverse genetics’ to investigate these pathways. In reverse genetics, researchers start with a gene of interest and investigate the phenotypic effect of altering its activity; by contrast, in ‘forward genetics’ the starting point is to look for a particular phenotype and then hunt out the altered gene that is causing it. Two reverse genetics approaches will be used by the consortium. Gene expression will be transiently knocked down with morpholinos, short segments of the gene that block its function. In addition, a recently developed technique known as TILLING (targeting induced local lesions in genomes) will be used to knock out gene activity permanently.The first step in TILLING is to mutagenise male zebrafish and mate them with untreated female fish, explains Stemple, whose group is one of three ZF-MODELS partners who will use this approach. Offspring are raised to adulthood, and the DNA of each individual is then genotyped for the exon of interest. The consortium already has a collection of 6,000 such individuals, and once a fish carrying a mutation in the gene of interest has been identified, it will be outcrossed to produce offspring, half of which will carry the desired mutation on one of their chromosomes. ‘It is then a matter of identifying these heterozygote fish and incrossing them to get homozygous fish in which you can see the phenotype that correlates with that mutation’, says Stemple.As well as helping to produce knock-outs for other researchers, Stemple is also using the TILLING technique to develop zebrafish models for muscular dystrophy. Among the genes that are important in notochord development are those that encode laminins. This led Stemple into studying muscular dystrophy because laminins are involved in the human disease. ‘When we used morpholinos to disrupt [the production of] dystroglycan, a laminin receptor, we got a good model for muscular dystrophy’, he explains. Now, he plans to use TILLING to disrupt up to 30 other genes known to be involved in human muscular dystrophy. ‘In particular, we will look for hypomorphic mutants, fish that are viable but on the edge of falling apart’. These mutants can be used to identify small molecules that push the fish into muscular dystrophy. Finding molecules that can cause a disease in this way ‘might give us a handle on something to fix the disease’, says Stemple.In another strand of the ZF-MODELS project, zebrafish expressing green fluorescent protein (GFP) in specific cells or tissues will be generated and characterised ( Figure 3 ). In such fish, developing structures can be easily imaged over time in the living embryo. One researcher working on this aspect of the project is Stephen Wilson, Professor of Developmental Genetics at University College London (United Kingdom). GFP lines can be made either by attaching to the GFP gene regions of DNA that control, or ‘drive’, GFP expression in selected cell types or by allowing the GFP gene to insert randomly in the genome and looking for fish with specific expression patterns. ‘There are now many lines of fish available with different GFP expression patterns’, says Wilson, ‘and it is important to catalogue their expression so that people can use the most appropriate lines for their research’.Wilson's own interest is in neuroanatomy. Together with Jon Clarke, another developmental neurobiology group leader at University College London, he plans to analyse GFP lines in which small groups of neurons or particular parts of neurons are labelled, and in this way start to build a detailed reconstruction of early brain neuroanatomy. This, in combination with other work on zebrafish carrying mutations affecting neural development, will give the team ‘a better picture of how a vertebrate brain is built’.A final, important aspect of the ZF-MODELS project, adds Dahm, is database construction. ‘We will be developing a set of databases that will integrate all of the project data’, he explains. ‘In addition, we hope to integrate our data with that of ZFIN in the United States to make one central zebrafish resource’.But Fish Aren't People The researchers of the ZF-MODELS consortium are understandably excited about participating in what will, says Geisler, bring an already strong European zebrafish community closer together. But zebrafish researchers in the United States are also excited by the ZF-MODELS project. ‘We need big lab models like ZF-MODELS in developmental biology’, says Hopkins, noting that the days of small groups working in isolation are long gone. This consortium, adds Howard Hughes Medical Institute Investigator Leonard Zon (Harvard Medical School, Boston, Massachusetts, United States), ‘will not only have an effect on European zebrafish science but also on how it is done in the United States’. But how much can zebrafish tell us about human development and disease? A lot, say zebrafish researchers. ‘Fish really are just little people with fins’, says Hopkins. ‘Of course, there are developmental differences between people and fish, and no one pretends that we can answer every question about human development in zebrafish’. Nevertheless, zebrafish studies can provide valuable clues to the genes involved in human diseases and to potential targets for therapeutic interventions. Hopkins provides the following illustration: ‘We have been doing “shelf screens”, in which we go back to our collection of mutants to find all those that affect the development of a single organ. When Zhaoxia Sun, a postdoc in my lab who now has an independent position at Yale Medical School, screened three-day-old fish for cystic kidney disease, she found 12 different genes. Two were known to cause human cystic kidney disease, so we knew we were in the human disease pathway somewhere, but we had no idea what the other genes were’. Hopkins and Sun have since identified the remaining genes, and these point to a single pathway being involved in the human disease. Developmental geneticist Didier Stainier (University of California, San Francisco, California, United States) is also using zebrafish to study organ development, in particular, heart development. The zebrafish heart is like the early human heart—a tube with an atrium, ventricle, and valves. ‘Everything we have found in the fish is relevant to the human heart’, says Stainier. ‘Obviously, there are additional processes involved in humans, but the basic outline of heart development in fish and people is largely similar’. Stainier has a collection of zebrafish in which valve formation is faulty. ‘Some of the genes we have found will be involved in human congenital valve defects’, he predicts. Knowing the identity of these genes will be useful diagnostically, but, in addition, zebrafish studies can reveal exactly what has gone wrong at a cellular level. The ability to follow individual cells as organs develop is key to this, says Stainier, who reported in March that fibronectin is required for heart development because, by regulating the polarisation of epithelial cells, fibronectin ensures the correct migration of myocardial cells. And in this issue of PLoS Biology, Stainier's lab have identified another zebrafish gene that is involved in heart development—cardiofunk, which encodes a special type of muscle protein.
But Fish Aren't People
The researchers of the ZF-MODELS consortium are understandably excited about participating in what will, says Geisler, bring an already strong European zebrafish community closer together. But zebrafish researchers in the United States are also excited by the ZF-MODELS project. ‘We need big lab models like ZF-MODELS in developmental biology’, says Hopkins, noting that the days of small groups working in isolation are long gone. This consortium, adds Howard Hughes Medical Institute Investigator Leonard Zon (Harvard Medical School, Boston, Massachusetts, United States), ‘will not only have an effect on European zebrafish science but also on how it is done in the United States’.But how much can zebrafish tell us about human development and disease? A lot, say zebrafish researchers. ‘Fish really are just little people with fins’, says Hopkins. ‘Of course, there are developmental differences between people and fish, and no one pretends that we can answer every question about human development in zebrafish’. Nevertheless, zebrafish studies can provide valuable clues to the genes involved in human diseases and to potential targets for therapeutic interventions. Hopkins provides the following illustration: ‘We have been doing “shelf screens”, in which we go back to our collection of mutants to find all those that affect the development of a single organ. When Zhaoxia Sun, a postdoc in my lab who now has an independent position at Yale Medical School, screened three-day-old fish for cystic kidney disease, she found 12 different genes. Two were known to cause human cystic kidney disease, so we knew we were in the human disease pathway somewhere, but we had no idea what the other genes were’. Hopkins and Sun have since identified the remaining genes, and these point to a single pathway being involved in the human disease.Developmental geneticist Didier Stainier (University of California, San Francisco, California, United States) is also using zebrafish to study organ development, in particular, heart development. The zebrafish heart is like the early human heart—a tube with an atrium, ventricle, and valves. ‘Everything we have found in the fish is relevant to the human heart’, says Stainier. ‘Obviously, there are additional processes involved in humans, but the basic outline of heart development in fish and people is largely similar’. Stainier has a collection of zebrafish in which valve formation is faulty. ‘Some of the genes we have found will be involved in human congenital valve defects’, he predicts. Knowing the identity of these genes will be useful diagnostically, but, in addition, zebrafish studies can reveal exactly what has gone wrong at a cellular level. The ability to follow individual cells as organs develop is key to this, says Stainier, who reported in March that fibronectin is required for heart development because, by regulating the polarisation of epithelial cells, fibronectin ensures the correct migration of myocardial cells. And in this issue of PLoS Biology, Stainier's lab have identified another zebrafish gene that is involved in heart development—cardiofunk, which encodes a special type of muscle protein.A Proliferation of Zebrafish Models of Human Disease Many researchers are now recognising the value of zebrafish models of human disease. Over the past three to four years, says Zon, this area of research has become a growth industry. The interest in disease models has grown hand-in-hand with the development of morpholinos to knock out specific genes, and the advent of TILLING, says Zon, ‘has set off a whole new fury. There are now large numbers of investigators who will try to knock out their favourite gene and come up with a model’. Zon has worked on disease models for blood ( Figure 4 ), blood vessel, and heart disorders but is currently studying zebrafish models of cancer. ‘We started by doing chemical mutagenesis and screened for cell-cycle mutants. These were embryonic lethals, but when we looked at heterozygote carriers of these mutations, some developed cancer at a high rate as adults’. Now Zon and his colleagues have returned to the cell-cycle mutant that yielded this cancer-susceptible heterozygote and are using embryos in high-throughput screening assays to look for small molecules that can suppress the cell-cycle phenotype. These molecules, reasons Zon, may have potential as anticancer drugs. Figure 4 Zebrafish kugelig Mutants The image shows live one-week-old zebrafish embryos. The embryos around the outside are wild-type fish. Those in the middle are a mutant called  kugelig  and have a homozygous mutation in a gene called  cdx4 . Loss of the proper functioning of this gene causes the obvious trunk and tail defects but also causes a reduction in the number of haematopoietic stem cells in the embryos, which therefore become severely anaemic. Studies on this mutant might lead to the discovery of molecules that can drive stem cell differentiation, for example, or could help improve understanding of human haematological malignancies. (Image courtesy of Alan Davidson, Harvard Medical School, Boston, Massachusetts, United States.)
A Proliferation of Zebrafish Models of Human Disease
Many researchers are now recognising the value of zebrafish models of human disease. Over the past three to four years, says Zon, this area of research has become a growth industry. The interest in disease models has grown hand-in-hand with the development of morpholinos to knock out specific genes, and the advent of TILLING, says Zon, ‘has set off a whole new fury. There are now large numbers of investigators who will try to knock out their favourite gene and come up with a model’.Zon has worked on disease models for blood ( Figure 4 ), blood vessel, and heart disorders but is currently studying zebrafish models of cancer. ‘We started by doing chemical mutagenesis and screened for cell-cycle mutants. These were embryonic lethals, but when we looked at heterozygote carriers of these mutations, some developed cancer at a high rate as adults’. Now Zon and his colleagues have returned to the cell-cycle mutant that yielded this cancer-susceptible heterozygote and are using embryos in high-throughput screening assays to look for small molecules that can suppress the cell-cycle phenotype. These molecules, reasons Zon, may have potential as anticancer drugs.And the Future of Zebrafish Research? Bigger and bigger seems to be the consensus. Chemical screens like Zon's for anticancer drugs can be set up for other human diseases such as muscular dystrophy. Work like Stainier's on organ development may have applications in tissue engineering. ‘If we can find out what drives differentiation in zebrafish’, he suggests, ‘we might be able to do the same for human cells’, making human tissue replacement therapy a practical possibility. And while many zebrafish researchers will continue to study development, others are now moving into the realms of physiology and behavioural studies. Geisler sums up zebrafish developmental research past, present, and future as follows: ‘No other [vertebrate] organism offers the same combination of transparent and accessible embryos, cost-effective mutagenesis screening, and, more recently, a sequenced genome, [DNA] chip, GFP, and knockout technology’. Add to that the potential of zebrafish embryos as a screening platform for small molecule libraries and the new technologies that allow forward and reverse genetics, and it is clear that zebrafish are not about to revert to being pretty pets swimming in small tanks in the corner of the living room.
And the Future of Zebrafish Research?
Bigger and bigger seems to be the consensus. Chemical screens like Zon's for anticancer drugs can be set up for other human diseases such as muscular dystrophy. Work like Stainier's on organ development may have applications in tissue engineering. ‘If we can find out what drives differentiation in zebrafish’, he suggests, ‘we might be able to do the same for human cells’, making human tissue replacement therapy a practical possibility. And while many zebrafish researchers will continue to study development, others are now moving into the realms of physiology and behavioural studies.Geisler sums up zebrafish developmental research past, present, and future as follows: ‘No other [vertebrate] organism offers the same combination of transparent and accessible embryos, cost-effective mutagenesis screening, and, more recently, a sequenced genome, [DNA] chip, GFP, and knockout technology’. Add to that the potential of zebrafish embryos as a screening platform for small molecule libraries and the new technologies that allow forward and reverse genetics, and it is clear that zebrafish are not about to revert to being pretty pets swimming in small tanks in the corner of the living room.Where to Find Out More ZF-MODELS More details of the work included in this European Union Integrated Project can be found at  http://www.zf-models.org
 ZFIN The ZFIN Web site, at  http://z.n.org/ZFIN , provides an extensive database for the zebrafish community including genetic, genomic, and developmental information; search engines for zebrafish researchers and laboratories; listings of meetings; and links to many other zebrafish sites, including sites with movies of zebrafish development. The special issue of  Development  (Dec 1; 1996; 123: 1–461) on the first two mutagenesis screens contains 37 research articles and can be freely accessed at  http://dev.biologists.org/content/vol123/issue1/index.shtml
Where to Find Out More
ZF-MODELSMore details of the work included in this European Union Integrated Project can be found at  http://www.zf-models.orgZFINThe ZFIN Web site, at  http://z.n.org/ZFIN , provides an extensive database for the zebrafish community including genetic, genomic, and developmental information; search engines for zebrafish researchers and laboratories; listings of meetings; and links to many other zebrafish sites, including sites with movies of zebrafish development.The special issue of  Development  (Dec 1; 1996; 123: 1–461) on the first two mutagenesis screens contains 37 research articles and can be freely accessed at  http://dev.biologists.org/content/vol123/issue1/index.shtmlDevelopment


Background Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model.
Background
Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model.Methods A suspension of 5 × 10 6  RCN-9 (rat colon cancer cells) was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522 (0, 3, 10, or 30 mg/kg/day) was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF.
Methods
A suspension of 5 × 10 6  RCN-9 (rat colon cancer cells) was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522 (0, 3, 10, or 30 mg/kg/day) was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF.Results JTE-522 dose-dependently decreased lung weight (p = 0.001) and the size of pulmonary metastatic tumors (p = 0.0002). However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors.
Results
JTE-522 dose-dependently decreased lung weight (p = 0.001) and the size of pulmonary metastatic tumors (p = 0.0002). However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors.Conclusion JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer.
Conclusion
JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer.

Background Colorectal cancer is a widespread fatal malignancy in the West [ 1 ] and its incidence in Japan is increasing [ 2 ]. Epidemiological studies have shown that regular doses of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with lower rates of colorectal polyps, colorectal cancer, and colorectal cancer-associated mortality. Waddell and Loughry initially reported that sulindac decreased the number of polyps in patients with familial adenomatous polyposis (FAP) [ 3 ]. The largest study of aspirin and colon cancer was undertaken by Thun  et al . in 1991 [ 4 ]. They found that colon cancer mortality decreased among those who used aspirin, and that the protective effect was increased by increasing doses. NSAIDs inhibit prostaglandin production mainly by inhibiting cyclooxygenase (COX) that is expressed as COX-1 and COX-2 isoforms. Cox is a key enzyme, which converts arachidonic acid to prostaglandins. COX-1 is constitutively expressed in most tissues and is thought to be responsible for homeostatic functions. On the other hand, COX-2 expression is induced by various factors including cytokines, growth factors, and tumor promoters [ 5 , 6 ]. Many studies have indicated that COX-2 expression is associated with both the carcinogenesis and growth of colon cancer. COX-2 is overexpressed in 85% of human colorectal cancers [ 7 ]. Oshima  et al . disclosed that COX-2 gene knockouts and a COX-2 inhibitor decreased the number of polyps of apcΔ716 knockout mice [ 8 ]. In addition, several studies have shown that the selective inhibition of COX-2 reduces colorectal tumorigenesis in various models of carcinogenesis. Reddy  et al . found that the COX-2 inhibitor, SC-58635 (celecoxib), significantly suppressed colonic aberrant crypt foci and crypt multiplicity in rats [ 9 ]. This supports the hypothesis that COX-2 inhibitors are chemopreventive. Kawamori  et al . showed that celecoxib inhibited both the incidence and multiplicity of colon tumors in a rat model [ 10 ], and another study showed that COX-2 expression in colon cancer cells increased metastatic potential [ 11 ]. We reported that increased COX-2 levels in human colorectal carcinomas correlated with both the size and invasiveness of tumors [ 12 ]. Tomozawa  et al . demonstrated a correlation between COX-2 overexpression and the recurrence of colorectal cancer [ 13 ]. It is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung [ 14 ]. A laparotomy is necessary for making experimental model of liver metastasis in rats, but it is also a stressful event. On the other hand, it is less invasive to make lung metastasis by injection of tumor cells into the tail vein of rats. Thus, we considered that the experimental model of pulmonary metastasis was appropriate for the evaluation of the real effect of JTE-522, a selective COX-2 inhibitor, on hematogenous metastasis of colon cancer in rats. The aim of this study was to examine the effect of JTE-522 on hematogenous metastasis of colon cancer in a rat model.
Background
Colorectal cancer is a widespread fatal malignancy in the West [ 1 ] and its incidence in Japan is increasing [ 2 ]. Epidemiological studies have shown that regular doses of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with lower rates of colorectal polyps, colorectal cancer, and colorectal cancer-associated mortality. Waddell and Loughry initially reported that sulindac decreased the number of polyps in patients with familial adenomatous polyposis (FAP) [ 3 ]. The largest study of aspirin and colon cancer was undertaken by Thun  et al . in 1991 [ 4 ]. They found that colon cancer mortality decreased among those who used aspirin, and that the protective effect was increased by increasing doses. NSAIDs inhibit prostaglandin production mainly by inhibiting cyclooxygenase (COX) that is expressed as COX-1 and COX-2 isoforms. Cox is a key enzyme, which converts arachidonic acid to prostaglandins. COX-1 is constitutively expressed in most tissues and is thought to be responsible for homeostatic functions. On the other hand, COX-2 expression is induced by various factors including cytokines, growth factors, and tumor promoters [ 5 , 6 ]. Many studies have indicated that COX-2 expression is associated with both the carcinogenesis and growth of colon cancer. COX-2 is overexpressed in 85% of human colorectal cancers [ 7 ]. Oshima  et al . disclosed that COX-2 gene knockouts and a COX-2 inhibitor decreased the number of polyps of apcΔ716 knockout mice [ 8 ]. In addition, several studies have shown that the selective inhibition of COX-2 reduces colorectal tumorigenesis in various models of carcinogenesis. Reddy  et al . found that the COX-2 inhibitor, SC-58635 (celecoxib), significantly suppressed colonic aberrant crypt foci and crypt multiplicity in rats [ 9 ]. This supports the hypothesis that COX-2 inhibitors are chemopreventive. Kawamori  et al . showed that celecoxib inhibited both the incidence and multiplicity of colon tumors in a rat model [ 10 ], and another study showed that COX-2 expression in colon cancer cells increased metastatic potential [ 11 ]. We reported that increased COX-2 levels in human colorectal carcinomas correlated with both the size and invasiveness of tumors [ 12 ]. Tomozawa  et al . demonstrated a correlation between COX-2 overexpression and the recurrence of colorectal cancer [ 13 ].et alet alet alet alet alIt is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung [ 14 ]. A laparotomy is necessary for making experimental model of liver metastasis in rats, but it is also a stressful event. On the other hand, it is less invasive to make lung metastasis by injection of tumor cells into the tail vein of rats. Thus, we considered that the experimental model of pulmonary metastasis was appropriate for the evaluation of the real effect of JTE-522, a selective COX-2 inhibitor, on hematogenous metastasis of colon cancer in rats. The aim of this study was to examine the effect of JTE-522 on hematogenous metastasis of colon cancer in a rat model.Methods Preparation of JTE-522 The selective COX-2 inhibitor, JTE-522 [ 15 ], was suspended in 0.5% carboxymethyl cellulose sodium salt (Wako Life Science Reagents, Osaka, Japan). Animals Twenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan). Tumors The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue  et al . after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [ 16 ]. The tumor cells were stored at -80°C. After thawing, tumor cells were washed in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 0.05% penicillin-streptomycin (Sigma). After repeated subculture, cell viability was assessed by Trypan blue dye exclusion and the cells were counted using a hemocytometer. Induction of lung metastases F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10 6  RCN-9 cells in 0.4 ml of PBS was injected into the tail vein. Pulmonary metastases of colon cancer in rats Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide. Immunohistochemistry Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% H 2 O 2  in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI; dilution 1:250) for 2 hours or vascular endothelial growth factor (VEGF) (rabbit polyclonal, VEGF A-20: sc-152; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200) for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine ®  Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained. Evaluation of immunostaining The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa  et al  [ 17 ]. Both the number of positive cells and the intensity of staining were evaluated as follows. The number of positive cells: none, 0; focal (one third of cells stained), 1; multifocal (two third of cells stained), 2; and diffuse (most cells stained), 3. The intensity of staining: none, 0; mild (between 0 and 2), 1; and strong (clearly identified by × 40 magnification), 2. The score for distribution and intensity were added and graded as follows: 0, 1, and 2: negative; and 3, 4, and 5: positive. Statistical analysis The effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05.
Methods
Preparation of JTE-522 The selective COX-2 inhibitor, JTE-522 [ 15 ], was suspended in 0.5% carboxymethyl cellulose sodium salt (Wako Life Science Reagents, Osaka, Japan).
Preparation of JTE-522
The selective COX-2 inhibitor, JTE-522 [ 15 ], was suspended in 0.5% carboxymethyl cellulose sodium salt (Wako Life Science Reagents, Osaka, Japan).Animals Twenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan).
Animals
Twenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan).Tumors The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue  et al . after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [ 16 ]. The tumor cells were stored at -80°C. After thawing, tumor cells were washed in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 0.05% penicillin-streptomycin (Sigma). After repeated subculture, cell viability was assessed by Trypan blue dye exclusion and the cells were counted using a hemocytometer.
Tumors
The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue  et al . after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [ 16 ]. The tumor cells were stored at -80°C. After thawing, tumor cells were washed in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 0.05% penicillin-streptomycin (Sigma). After repeated subculture, cell viability was assessed by Trypan blue dye exclusion and the cells were counted using a hemocytometer.et alInduction of lung metastases F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10 6  RCN-9 cells in 0.4 ml of PBS was injected into the tail vein.
Induction of lung metastases
F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10 6  RCN-9 cells in 0.4 ml of PBS was injected into the tail vein.Pulmonary metastases of colon cancer in rats Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide.
Pulmonary metastases of colon cancer in rats
Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide.Immunohistochemistry Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% H 2 O 2  in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI; dilution 1:250) for 2 hours or vascular endothelial growth factor (VEGF) (rabbit polyclonal, VEGF A-20: sc-152; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200) for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine ®  Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained.
Immunohistochemistry
Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% H 2 O 2  in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI; dilution 1:250) for 2 hours or vascular endothelial growth factor (VEGF) (rabbit polyclonal, VEGF A-20: sc-152; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200) for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine ®  Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained.Evaluation of immunostaining The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa  et al  [ 17 ]. Both the number of positive cells and the intensity of staining were evaluated as follows. The number of positive cells: none, 0; focal (one third of cells stained), 1; multifocal (two third of cells stained), 2; and diffuse (most cells stained), 3. The intensity of staining: none, 0; mild (between 0 and 2), 1; and strong (clearly identified by × 40 magnification), 2. The score for distribution and intensity were added and graded as follows: 0, 1, and 2: negative; and 3, 4, and 5: positive.
Evaluation of immunostaining
The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa  et al  [ 17 ]. Both the number of positive cells and the intensity of staining were evaluated as follows. The number of positive cells: none, 0; focal (one third of cells stained), 1; multifocal (two third of cells stained), 2; and diffuse (most cells stained), 3. The intensity of staining: none, 0; mild (between 0 and 2), 1; and strong (clearly identified by × 40 magnification), 2. The score for distribution and intensity were added and graded as follows: 0, 1, and 2: negative; and 3, 4, and 5: positive.et alStatistical analysis The effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05.
Statistical analysis
The effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05.Results Pulmonary metastases Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs (p = 0.0001, Figure  1A ). Cross sections contained many metastatic tumors that were not visible on the surface (Figures  2 ). The size of the metastatic tumors was significantly and dose-dependently decreased (p = 0.0002, Figure  1B ), although the number of metastases did not differ (Figure  1C ). JTE-522 appeared to have no significant adverse effects. Immunohistochemistry Immunohistochemical staining showed that both COX-2 and VEGF were expressed at high levels in pulmonary metastatic tumors of all groups (Figures  3 ). The large airways and vascular structures were stained for both proteins, as were tumor cells in all groups. However, there was no correlation between COX-2 score and VEGF score (p = 0.60, Figure  4 ). The degree of COX-2 and VEGF immunohistochemical staining in tumor cells did not significantly differ among groups (Table  1 ).
Results
Pulmonary metastases Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs (p = 0.0001, Figure  1A ). Cross sections contained many metastatic tumors that were not visible on the surface (Figures  2 ). The size of the metastatic tumors was significantly and dose-dependently decreased (p = 0.0002, Figure  1B ), although the number of metastases did not differ (Figure  1C ). JTE-522 appeared to have no significant adverse effects.
Pulmonary metastases
Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs (p = 0.0001, Figure  1A ). Cross sections contained many metastatic tumors that were not visible on the surface (Figures  2 ). The size of the metastatic tumors was significantly and dose-dependently decreased (p = 0.0002, Figure  1B ), although the number of metastases did not differ (Figure  1C ). JTE-522 appeared to have no significant adverse effects.Immunohistochemistry Immunohistochemical staining showed that both COX-2 and VEGF were expressed at high levels in pulmonary metastatic tumors of all groups (Figures  3 ). The large airways and vascular structures were stained for both proteins, as were tumor cells in all groups. However, there was no correlation between COX-2 score and VEGF score (p = 0.60, Figure  4 ). The degree of COX-2 and VEGF immunohistochemical staining in tumor cells did not significantly differ among groups (Table  1 ).
Immunohistochemistry
Immunohistochemical staining showed that both COX-2 and VEGF were expressed at high levels in pulmonary metastatic tumors of all groups (Figures  3 ). The large airways and vascular structures were stained for both proteins, as were tumor cells in all groups. However, there was no correlation between COX-2 score and VEGF score (p = 0.60, Figure  4 ). The degree of COX-2 and VEGF immunohistochemical staining in tumor cells did not significantly differ among groups (Table  1 ).Discussion JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer  et al . described the inhibition of tumor growth and lung metastasis of Lewis Lung carcinoma in mice by continuous dietary celecoxib (160–3200 ppm) supplied from the date of implantation [ 18 ]. Celecoxib at a dose of 160 ppm did not affect the number and size of lung metastases, but doses between 480 and 3200 ppm reduced the number of metastatic nodules by >50%. Histopathological analysis revealed that celecoxib dose-dependently reduced the size of the metastatic lung tumors. Masferrer  et al . evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa  et al . investigated the effect of JTE-522 on lung metastasis of colon cancer in mice [ 19 ]. Their study showed that JTE-522 significantly reduced the number of metastatic nodules and lung weight. They macroscopically counted the metastatic tumors on the lung surface. Our study using cross sections disclosed that many nodules were not visible on the surface and could not be seen by the naked eye. These observations may indicate that JTE-522 blocks the growth of metastatic tumors, but does not inhibit the process of metastasis. On the other hand, a recent study in vitro, using cancer cell lines preincubated with celecoxib, suggests that COX-2 is involved in adhesion of cancer cells to vascular endothelium [ 20 ]. If the cancer cells had been pretreated with JTE-522 for an appropriate period in the present study, it might reduce the number of the lung metastases. Here, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression [ 21 , 22 ] and that COX-2 inhibitors directly affect angiogenesis [ 23 ]. Other recent studies have confirmed the notion that tumor growth and metastasis are dependent on angiogenesis and require the development of new vessels [ 24 - 26 ], and that COX-2 is related to tumor angiogenesis. COX-2 modulates the production of angiogenic factors derived from colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells [ 27 ]. COX-2 is also related to tumor angiogenesis in human colorectal cancer. Cianchi  et al . identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway [ 28 ]. Immunohistochemical staining in the present study revealed parallel COX-2 and VEGF expression that was mainly localized in the cytoplasm of tumor cells, epithelial cells of the airways, and vascular structures. These findings may indicate that COX-2 is involved in the production of VEGF. Furthermore, the present study supports our previous report that JTE-522 interferes with the growth of metastatic tumors of colon cancer, due to inhibition of neovascularization [ 29 ]. Our results showed that high levels of COX-2 were expressed in the groups administered with JTE-522 as well as in controls. This is consistent with the fact that JTE-522 suppresses not the production, but the activity of COX-2 in prostaglandin biosynthesis [ 15 ]. VEGF was also highly expressed in all groups. One of the reasons may be hypoxia induced by multiple pulmonary metastases. Christou  et al . reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls [ 30 ]. On the other hand, these findings may indicate that VEGF is produced via a pathway, which does not include COX-2, as well as through a COX-2 pathway. We reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters [ 31 ]. Additionally, COX-2 expression and degree of dysplasia are significantly associated in adenomas [ 32 ]. COX-2 levels in human colorectal carcinomas are significantly higher in larger tumors and in those that invade more deeply [ 12 ]. We therefore postulated that COX-2 might correlate with metastasis as well as primary colorectal tumor, because hematogenous metastasis is the key for prognostic factor in colorectal cancer. The advances in the treatment for metastasis would improve the prognosis of the patients with colorectal cancer. The present study found that JTE-522 dose-dependently suppressed metastatic tumor growth, although the function to prevent cancer cells from metastasizing might not be so strong. Our recent report has revealed that administration of rofecoxib, a selective COX-2 inhibitor, decreased the number and size of rectal polyps in familial adenomatous polyposis patients, whereas the relapse of rectal polyps was seen 3 months after completion of the treatment [ 33 ]. This phenomenon might indicate that COX-2 inhibitors make colorectal polyps invisible, although atypical cells still exist microscopically. This is consistent with the result of the present study. Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao  et al . demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms [ 34 ]. Fenwick  et al . reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases [ 35 ]. It is considered that COX-2 inhibitors can be used as therapeutic agents for hematogenous metastases of colorectal cancer.
Discussion
JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer  et al . described the inhibition of tumor growth and lung metastasis of Lewis Lung carcinoma in mice by continuous dietary celecoxib (160–3200 ppm) supplied from the date of implantation [ 18 ]. Celecoxib at a dose of 160 ppm did not affect the number and size of lung metastases, but doses between 480 and 3200 ppm reduced the number of metastatic nodules by >50%. Histopathological analysis revealed that celecoxib dose-dependently reduced the size of the metastatic lung tumors. Masferrer  et al . evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa  et al . investigated the effect of JTE-522 on lung metastasis of colon cancer in mice [ 19 ]. Their study showed that JTE-522 significantly reduced the number of metastatic nodules and lung weight. They macroscopically counted the metastatic tumors on the lung surface. Our study using cross sections disclosed that many nodules were not visible on the surface and could not be seen by the naked eye. These observations may indicate that JTE-522 blocks the growth of metastatic tumors, but does not inhibit the process of metastasis. On the other hand, a recent study in vitro, using cancer cell lines preincubated with celecoxib, suggests that COX-2 is involved in adhesion of cancer cells to vascular endothelium [ 20 ]. If the cancer cells had been pretreated with JTE-522 for an appropriate period in the present study, it might reduce the number of the lung metastases.et alet alet alHere, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression [ 21 , 22 ] and that COX-2 inhibitors directly affect angiogenesis [ 23 ]. Other recent studies have confirmed the notion that tumor growth and metastasis are dependent on angiogenesis and require the development of new vessels [ 24 - 26 ], and that COX-2 is related to tumor angiogenesis. COX-2 modulates the production of angiogenic factors derived from colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells [ 27 ]. COX-2 is also related to tumor angiogenesis in human colorectal cancer. Cianchi  et al . identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway [ 28 ]. Immunohistochemical staining in the present study revealed parallel COX-2 and VEGF expression that was mainly localized in the cytoplasm of tumor cells, epithelial cells of the airways, and vascular structures. These findings may indicate that COX-2 is involved in the production of VEGF. Furthermore, the present study supports our previous report that JTE-522 interferes with the growth of metastatic tumors of colon cancer, due to inhibition of neovascularization [ 29 ]. Our results showed that high levels of COX-2 were expressed in the groups administered with JTE-522 as well as in controls. This is consistent with the fact that JTE-522 suppresses not the production, but the activity of COX-2 in prostaglandin biosynthesis [ 15 ]. VEGF was also highly expressed in all groups. One of the reasons may be hypoxia induced by multiple pulmonary metastases. Christou  et al . reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls [ 30 ]. On the other hand, these findings may indicate that VEGF is produced via a pathway, which does not include COX-2, as well as through a COX-2 pathway.et alet alWe reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters [ 31 ]. Additionally, COX-2 expression and degree of dysplasia are significantly associated in adenomas [ 32 ]. COX-2 levels in human colorectal carcinomas are significantly higher in larger tumors and in those that invade more deeply [ 12 ]. We therefore postulated that COX-2 might correlate with metastasis as well as primary colorectal tumor, because hematogenous metastasis is the key for prognostic factor in colorectal cancer. The advances in the treatment for metastasis would improve the prognosis of the patients with colorectal cancer. The present study found that JTE-522 dose-dependently suppressed metastatic tumor growth, although the function to prevent cancer cells from metastasizing might not be so strong. Our recent report has revealed that administration of rofecoxib, a selective COX-2 inhibitor, decreased the number and size of rectal polyps in familial adenomatous polyposis patients, whereas the relapse of rectal polyps was seen 3 months after completion of the treatment [ 33 ]. This phenomenon might indicate that COX-2 inhibitors make colorectal polyps invisible, although atypical cells still exist microscopically. This is consistent with the result of the present study.Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao  et al . demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms [ 34 ]. Fenwick  et al . reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases [ 35 ]. It is considered that COX-2 inhibitors can be used as therapeutic agents for hematogenous metastases of colorectal cancer.et alet alConclusion In conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer.
Conclusion
In conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its coordination. All authors read and approved the final manuscript.
Authors' contributions
HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its coordination. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: