Pro-Test Blogs!

Brain metastasis that affect at least 20% of cancer patients are a serious problem for doctors seeking to treat cancer and kill thousands of patients every year, being particularly difficult to treat because many anti-cancer drugs cannot cross the blood-brain barrier and because surgery to remove the tumor can often be difficult and risky. Patients suffering from breast, lung and skin cancer run a relatively high risk that their cancer will spread to the brain, a worrying fact considering that these are amongst the most common of cancers . As a consequence of this scientists are very keen to understand how cancer spreads to the brain, with the ultimate aim of preventing that spread.

It has long been thought that brain metastasis is due to interactions between cells that are shed by the primary tumor and the nerve cells of the brain, but real evidence of this from living animals and humans for that theory has been hard to find, and in recent years observations made in animal models of cancer have suggested that blood vessels in the brain rather than nerve cells are the site of the early growth of tumor cells during brain metastasis. This week a paper in the open access journal PLoS One reports on work done by scientists at Oxford University that confirms that during brain metastasis tumor cells do indeed bind to blood vessels and form tumors before spreading into the surrounding nerve tissue, a result of huge importance to the future treatment and prevention of brain metastasis.

To demonstrate this Dr Shawn Carbonel and colleagues (1) injected breast and skin tumor cells into the bloodstream or fat tissue of mice and then after several days humanely killed the mice determined where in the brain the micrometastases, small colonies of tumor cells that later grow into tumors, were forming, and found that almost all were associated with the blood vessels. There was no sign of any new blood vessel growth, which indicated that the metastases were associating with the blood vessels, and that it wasn't simply the case that they were promoting the growth of new blood vessels in the vicinity of the growing tumor. To confirm that this is also true in humans they examined tissue samples that been donated following neurosurgery or autopsy and found that almost all metastases were associated with blood vessels, a finding that supported the results of their experiments in mice.

Now they had to answer a new question; were the micrometastases associated with the blood vessels because they have a preference for interacting with the cells of the blood vessel, or simply because the first part of the brain they come to is that adjecent to the blood vessel? To answer this the Oxford scientists injected tumor cells that were labelled with green fluorescent protein directly into an area of the brain allowing equal access to both blood vessels and nerve cells, and using a cranial window in the skulls of the mice were able to observe where the GFP-labelled tumor cells ended up. They observed that the GLF-labelled cells associated almost exclusively with blood vessels, and that the tumors subsequently grow into the surrounding brain tissue.

The tumor cells bind to a blood vessel structure called the vascular basement membrane (VBM), but what the Oxford scientists really wanted to know was what caused the tumor cells to bind to the VBM. Once again using mice with cranial windows fitted they found that an enzyme named focal adhesion kinase was highly active where the tumor cells were interacting with the VBM. Focal adhesion kinase is part of a pathway through which a class of proteins known as the integrins control the interaction between many cells and either other cells or extracellular proteins such as the components of the vascular basement membrane, an observation which suggested that an integrin plays a key role in the binding of tumor cells to the VBM. They next found that a particular integren named Beta 1 integrin is present on all the tumor cell lines they were studying, and that antibodies blocking it could prevent the tumor cells from binding components of the VBM in vitro and to blood vessels in human brain tissue slices.

But would the anti-Beta 1 integrin blocking antibody prevent tumor metastasis in living animals? The answer was yes, the antibodies greatly reduced the growth tumors from human breast tumor cells that were injected directly into the brains of mice. To further emphasize the importance of Beta 1 integrin in brain metastasis they found that when mouse lymphoma cells that had been genetically engineered to lack Beta 1 integrin were injected into mouse brains they formed far smaller tumors than non-GM lymphoma cells.
This study changes the way we look at the process of brain metastasis, and more importantly in Beta 1 integrin it identifies a target for new drugs, perhaps monoclonal antibodies, that block the binding of tumor cells to blood vessels and prevent brain metastasis. With this in mind it is useful to note that studies in mice have found that while Beta 1 integrin is crucial during embryonic development prolonged anti-Beta 1 therapy in adult animals did not produce any overt evidence of toxicity (2), indicating that it should also be possible to inhibit it safely in human patients during anti-cancer chemotherapy.

It's a very nice paper, my only gripe being that they didn't examine if anti-Beta 1 integrin blocking antibody therapy could prevent tumor cells injected into the mouse bloodstream from producing micrometastases in the blood vessels of the brain rather than just looking at the growth of tumor cells injected directly into the brain, though I expect that those experiments are now being done and will soon be reported. There will certainly be a lot of interest in this paper in the cancer research world, and scientists will seek to reproduce these results (a vital part of the scientific process) and then expand on them with their own studies of the safety and efficacy of this approach before clinical trials in humans can begin.

Regards

Paul Browne

1) Carbonell W.S. et al. "The vascular basement membrane as "soil" in brain metastasis." PLoS ONE Volume 4(6):e5857 (2009) DOI:10.1371/journal.pone.0005857

2) Park C.C. et al. "Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo."Cancer Res. Volume 66(3), Pages 1526-1535 (2006) DOI:10.1158/0008-5472.CAN-05-3071

If you watched the news or picked up a newspaper yesterday you'll already be aware that scientists in Japan have created genetically modified (GM) marmoset monkeys that pass the transgene, in this case one that encodes the marker GFP protein that glows under UV light, to their offspring. The Times, Guardian, Independent and BBC all report this advance in...err..glowing terms, and in the Times today the columnist Hugo Rifkind contrasts the scientists with animal rights activists who are "...prepared to sacrifice other people for monkeys". On this Hugo has a good point, arguments frequently rage over the question of animal rights supporters using medicines developed through animal research, but the real issue is not whether or not they use these medicines themselves but that they seek to deny them to others who do not share their views.

It's worth stressing that these are not the first GM monkeys, in 2001 scientists lead by Dr. Anthony Chan at the Oregon Regional Primate Research Centre produced the world's first transgenic monkey, a rhesus macaque named ANDi, and last year produced the first monkey model of Huntington's disease but none of these GM macaques have transferred the transgene to their offspring, possibly because it is not present in tissues such as the sperm and egg.

The group lead by Dr. Erika Sasaki (1) used a different technique to previous groups that have created transgenic monkeys, rather than introducing the transgene into unfertilized eggs and then fertilizing them by IVF they found that it was more efficient to take eggs that had already been fertilized naturally and then introduce the transgene, and they carefully adjusted the conditions of the transfer so that the maximum number of copies of the transgene reached the cells of the embryo. They chose the marmoset rather than the macaque for this study because it has a shorter life cycle than the macaque, which means that GM offspring can be generated much more quickly than with macaques, an advantage that means that it should be possible to establish colonies of GM marmosets for research far more quickly than would be the case for macaques. Dr. Sasaki and her colleagues expect that GM marmosets will become a valuable model for diseases where GM rodents are not able to provide all the information scientists require, such as amyotrophic lateral sclerosis and Huntington’s disease.

Against that macaques are closer to humans in evolutionary terms, so that some human diseases such as tuberculosis can be studied in macaques but not in marmosets, and the smaller brain size and lower cognitive ability of marmosets compared to macaques means that GM macaques will probably complement rather than replace macaques in neuroscience research. It is also probable that some of the techniques developed by Dr. Sasaki and her team can be used to improve the efficiency of GM macaque production, so this should be seen as a boost to GM monkey research in general.

So do these monkeys herald a "health revolution" as the Independent suggests? Well, perhaps evolution would be a more accurate term. Impressive as this achievement is more work will need to be done to improve it, especially to make sure that the correct number of transgenes are safely and efficiently delivered to the tissues where their expression is required. It is certainly worth remembering that while GM monkeys may become an important resource in tomorrow's medical research they will only ever account for a tiny fraction of GM animals, as they will be used only when scientists are unable to learn enough from GM rodents.

Paul Browne

1) Sasaki E. et al. "Generation of transgenic non-human primates with germline transmission" Nature Vol 459, pages 523-528 (2009) doi:10.1038/nature08090

A claim frequently made by animal rights activists is that by paying for animal research charities and other funding bodies are diverting money from other areas such as clinical research. However the reality is that clinical and animal scientists work together to understand what is going wrong in disease and to illuminate previously unknown aspects of biology. A good example of this process is provided by media reports that scientists studying the low incidence of solid cancerous tumours in people with Down's syndrome have discovered exciting new targets for the treatment and prevention of Cancer.

The team led by Dr Sandra Ryeom of the Children's Hospital Boston knew that among people with Down's syndrome the mortality rate from solid tumours is less than 10% of what would be expected, indicating that one or more of the 231 genes that people with Downs syndrome have an extra copy of as a result of the chromosome 21 trisomy (three copies rather than the normal two) is responsible. But which gene? From earlier in vitro studies they knew that large amounts of a protein called DSCR1 could block angiogenesis* by suppressing a pathway activated by the hormone vascular endothelial growth factor (VEGF) that is produced by many tumours. What they wanted to know was whether the relatively small increase in the level of Dscr1 seen in Down's syndrome could block angiogenesis, and to do this they turned to animal models where the interaction of tumour cells with surrounding tissues, including blood vessels, could be studied, and in particular to the Ts65Dn mouse model of Downs syndrome where the mice have an extra copy of 104 of the genes found in human Down's syndrome and exhibit many Down’s symptoms. In their paper published online in Nature (1) they describe how Dscr1 is one of the 104 extra genes and that when they transplanted two types of cancer cell into the mice they found that the tumours grew far more slowly in the Down's mice than in control mice. On examination tumours from Ts65Dn mice were found to contain fewer blood vessels than those from control mice.

They then performed further studies that demonstrated that the decrease in tumour growth was due to suppression of new blood vessel growth rather than interference with the tumours ability to hijack the blood supply in existing blood vessels. A question that remained was whether Dscr1 was the only extra gene involved, so they created a mouse model which has only the normal 2 copies Dscr11 but still has an extra copy of the 103 other genes. In this mouse model tumour growth was slower than in normal mice but faster than in the Ts65Dn mouse, suggesting that one extra copy of Dscr1 is necessary for maximal suppression of tumour growth via inhibition of tumour angiogenesis but that other genes are also involved. Further work showed that Dscr1 acts by decreasing the levels of the enzyme Cyclooxygenase 2, which is an important mediator of the angiogenic response to VEGF in the VEGF-calcineurin-NFAT pathway, and that another gene called Dyrk1a that is also present in an extra copy in Downs blocks the same VEGF-calcineurin-NFAT pathway, but by altering the function of NFAT.

Taken together with the clinical observations of the low levels of solid tumours in people with Down's syndrome these results emphasize how important angiogenesis is to the transition from non-cancerous micro-tumours to larger tumours that may eventually metastasize, and identify the VEGF-calcineurin-NFAT pathway as a promising target for the development of new anti-cancer drugs. It seems appropriate that the author list of this very interesting paper that will no doubt stimulate much research over the next few years includes Judah Folkman, a true pioneer in the field of angiogenesis who died last year.

* Angiogenesis is the growth of blood vessels and in cancer plays is important role in the growth of some tumours and their subsequent metastasis to other tissues in the body.

1) Baek K.-H. et al. "Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1" Nature, Advance online publication 20 May 2009, DOI:10.1038/nature08062;

Yesterday Members of the European Parliament (MEPs) voted by a overwhelming majority to approve a report submitted by the European Parliament's Agriculture Committee on proposed revisions to EU directive 86/609, the EU rules that ensure the protection of animals used for scientific purposes. The report approved by MEPs makes several welcome changes to earlier proposals for the long-awaited revision of EU directive 86/609 that were put forward by the EU Commission last year. We are delighted to learn that MEPs have listened to the many scientists, charities and patients who have discussed with them the potential impact of these revisions on scientific research with them over the course of the past few months and have asked them to remember the patients when they cast their vote. They clearly agree with Sir Terry Pratchett, who while speaking on behalf of Remember the Patients recently said:

"There's only two ways it can go: researchers, with as much help you can give them, may come up with something that reduces the effects of this dreadful, inhuman disease, or we will have to face the consequences of our failure to prevent the final years of many of us being a long bad dream."

MEPs have voted to remove articles that would have increased bureaucratic burden placed in scientists without improving the welfare of animals, for example by extending the scope of the directive to include hens eggs and microscopic crustaceans, and have also voted to amend an article that would have prevented monkeys from being used in important basic research that seeks to illuminate the processes that are involved in diseases such as Parkinson's and multiple sclerosis.

On hearing of the vote Professor Tipu Aziz, Oxford neuroscientist and Pro-Test science advisor said:

"With this vote MEPs are sending a clear message to the national governments that they want Europe to remain at the forefront of 21st century medical science, while also demonstrating their solidarity with the many patients who await the development of new treatments and cures"

While not perfect the proposed revisions to EU directive 86/609 that have been approved by the European parliament strike a good balance between encouraging and facilitating high quality medical research in Europe and protecting the welfare of the animals used in that research. They will now go back to the EU commission and then to the European Council of Ministers, and these bodies may either choose to accept the amendments made by the Parliament, at which point the revisions will become law, or reject and further amend some, which would trigger a further round of debate and voting. It is therefore crucial that the scientific community continues to engage with Europes politicians on this issue to safeguard the progress that has been achieved.

Pro-Test welcomes this vote and thanks the MEPs for voting to support and protect the future of medical research in Europe. Without such research medical advances for diseases such as Alzheimer’s and Parkinson’s would be at a huge disadvantage.

Looking closer to home we are pleased to see that Oxford University has published the number of primates it uses for animal experiments, but it is a pity that it took a Freedom of Information request by the BUAV to compel them to do so. Universities and other institutions that conduct animal research should strive to be as open and transparent as possible about the work that their scientists do, and while we acknowledge that there is still a need to protect staff and scientists from animal rights extremists care must be taken to ensure that the necessary precautions do not stand in the way of efforts to improve communication and dialogue. Despite the threat from extremists Oxford University has made great strides towards increased openness in recent years, even inviting documentary makers to film in its laboratories, and we hope that with the publication of these statistics it will redouble these laudable efforts.

Cheers

Paul Browne

Pro-Test salutes the 800 academics, citizens and students who rallied yesterday to show support for scientists at the University of California, Los Angeles (UCLA) who have faced threats, harassment and arson from animal rights extremists. UCLA Pro-Test, an organisation founded by neuroscientist David Jentsch, and a number of other scientists who have also been the victims of animal rights extremism in California, made a decisive stand for animal research and sent a clear message to animal rights extremists that "enough is enough".

As the rally stretched close to a quarter of a mile long Jentsch remarked that "I hope this rally lessens the sense of helplessness and fear that has pervaded our community, We’re just not going to take the harassment anymore", while fellow neuroscientist Neurology Professor Claude Westerlain, who uses animals in his research on epilepsy, said he came to the rally to oppose the terror tactics that some of his colleagues have faced.

"Even the [activists] with good intentions are mistaken when they say there is no need for animal research. There is no way to do research on epilepsy without animals, and the suffering we relieve is enormous. Animal research saves lives. There is no medical progress without it."

Among those attending the rally was UCLA neuroscientist Dario Ringach, who announced in 2006 that he would give up his animal research if activists would stop harassing him and his family but is now a member of the UCLA Pro-Test committee.

"I came because I don't think people should have to face a choice between the security of their family and their research" he said "I came to defend academic research and academic freedom."

UK Pro-Test member, Tom Holder, who recently founded Pro-Test's sister organisation in the US, Speaking of Research, flew across to the US to assist with the rally. Speaking at the rally he announced The Pro-Test Petition which supports biomedical research and condemns violent tactics used to campaign against it.

Well done to everyone who joined the Pro-Test UCLA rally, and especially to those brave scientists who made it happen!

Things have also also been busy on this side of the pond.

First the good news from Cancer Research UK that deaths from breast cancer are at their lowest levels for 40 years, an achievement that can be attributed to both improvements in screening that allows tumours to be detected earlier and new treatments discovered through animal research.

Second, as you may have heard the European Union is currently engaged in a much needed overhaul of its rules on the use of animals in medical research, and at the beginning of May MEPs will debate the proposed revisions in a plenary session of the EU Parliament. This revision is an opportunity to shape the future of medical research in Europe, if the new rules are sensible they will safeguard animal welfare while encouraging and facilitating top class research, if not then they have the potential to stifle much vital research throughout the EU and turn Europe into a medical research backwater. So if you haven't done so already we strongly encourage you to write to your MEPs and remind them to Remember the Patients when they vote in two weeks time.