Tag Archives: tumor

Of Mice and Mammaries, Part 2: Breast cancer in a dish?

In light of Breast Cancer Awareness Month, Justin Varholick traces how mice have helped breast cancer research over the past century. In the second post of this 4-part series, we look at advances made from 1960 to 1975 when scientists were studying a virus in the milk.

Last week, in Part 1 of this series, we discussed how scientists from the early 1900s studied the growth and spread of mammary tumors in mice. We also walked through an experiment discovering how the breast milk from mother mice carried “something” that was more responsible for breast cancer than the genes the mother had passed down to her pups. This week we learn how scientists determined that this new “something” was a virus, and this virus could only be found in mice – it was never found in humans.

1940 to 1960 — What’s in the milk?

After Dr. John Bittner found out that there was “something” in the milk, many scientists wanted to figure out what it was. A first thought was to filter the milk and see if it still showed the same effects as Dr. Bittner’s experiments. Scientists took very special filters used to filter out bacteria, and ran mouse milk from “high tumor” mothers through these filters. Despite filtering, the results remained the same – filtered milk from “high tumor” mothers still led to tumors in pups fed the milk.

Then in the early 1950s scientists started using electron microscopes to compare “high-tumor” mouse breast milk with normal “low tumor” mouse breast milk. The electron microscopes gave scientists the power to magnify the milk 2,000,000x the normal size – magnification beyond what the bacteria filters could filter out. After comparing the types of milk, Dr. Leon Dmochowski found many small particles in the “high tumor” milk and very few of these particles in the “low tumor” milk. After other scientists repeated these experiments, they concluded that these particles were the “something” in the milk that may be responsible for tumors in the pups. Many scientists looked for these particles in human breast tissue and milk, but could never find it – only mice have this “something” in the milk. It was interesting, however, that the particles were in both “high tumor” mothers and “low tumor” mothers, albeit in different amounts, which indicated that both types of mothers were at risk to have the mammary tumors – “high tumor” mothers just had a higher risk.

While all this science with electron microscopy was going on, scientists studying mammary tumor cells in mice determined that the something in the milk acted very much like a virus and gave it the name mouse mammary tumor virus, or MMTV for short.

Cover of “Immunity against the mouse mammary tumor virus” by Paula Creemers. Electron microscope image of MMTV with a sketch of two mice with mammary tumors.

1960 to 1975 — Can we grow the milk virus in a dish?

Now that scientists had established that a virus in the milk was responsible for mammary tumors in mice, they wanted to see if they could grow the virus in a petri dish. Scientists had already grown viruses in a dish that were responsible for cancer in chickens, and other viruses that were responsible for skin cancer in humans, so they believed they would be able to grow MMTV in the dish. Unfortunately, growing the milk virus dish proved very difficult.

In the 1970s many scientists tried to grow the virus in a dish and were unsuccessful. One group of scientists at the Cancer Research Genetics Laboratory (CRGL) of the University of California, Berkeley showed that the virus could be grown in a dish, but it was too cumbersome for many scientists to use for research. Because MMTV could not be easily grown in a dish, scientists interested in mammary tumors in mice knew they had to find a new method if they wanted to continue using mice to understand more about breast cancer.

To be continued…

Tune in next week, to learn how scientists started using new methods with stem cells to make genetically engineered mice and how they validated that the mouse could be used as a model for humans!

Justin Varholick

References:

  1. Cardiff R, Kenney N. (2011). A compendium of the mouse mammary tumor biologist: From the initial observations in the house mouse to the development of genetically engineered mice. Cold Spring Harb Perspect Biol. 3(6).

 

En Passage, an Approach to the Use and Provenance of Immortalized Cell Lines

This guest post is by Lisa Krugner-Higby, DVM, PhD.  Dr. Krugner-Higby is a scientist and also a research veterinarian within the Research Animal Resource Center at the University of Wisconsin-Madison. Dr. Krugner-Higby’s research is in development of extended-release formulations of analgesic and antimicrobial drugs. She previously worked in anti-HIV drug development.

I am always fascinated by the idea promoted by some animal rights activists – repeated in many versions and for many decades – that all preclinical biomedical research can be conducted using in vitro cell culture. I have never found one of them who has spent much time working with cell culture. On the other hand, I have spent approximately seven years of my life working with cell cultures, looking at the stainless steel back wall of a laminar flow work station day after day. One thing I can say about immortalized cell lines, or cells that reproduce indefinitely, is that they are not alive in the same way that a mouse is alive.

 

Cell culture

Cell culture

The first thing that a graduate student learns when they begin to work with cell culture is how to take cells that have overgrown the sterile plastic flask they inhabit and put them into a fresh flask with fresh growth medium. It’s called ‘splitting’ the number of cells and ‘passaging’ them into a new home. Split and passage, split and passage… I knew that with every passage, the cell line became a little more different than normal cells and even a little more different than the original cell line. The remedy for this type of genetic drift was to freeze low passage cells in liquid nitrogen and re-order the line from the repository when the low passage stocks were depleted. I was careful with my sterile technique, cleaned the laminar flow hood, and used a new sterile pipet every time in order to avoid contamination of my cells. Unfortunately, the day came when I opened the incubator door and the flasks were black and fuzzy with fungus, and all of my carefully tended cells were dead. An anguished conversation with the tissue culture core technician revealed that this happened every Spring in North Carolina when the physical plant turned on the air conditioning for the year, blowing a Winter’s worth of fungal spores out of the ductwork and into the air. She recommended doing other things for about 6 weeks until the spore load had blown out of the ducts. I have had other cell line disasters in my scientific career: the malfunctioning incubator thermostat that turned a colleague’s two months’ worth of cell culture growth into flasks of overheated goo or that generally reputable vendor that sold us a case of tissue culture flasks that were not properly sterilized resulting in clouds of bacteria in the warm, moist, nutrient-rich environment of the incubator.

I never thought to ask, in those early days, if the cells that I fussed, worried, and wept over, were actually the cells that they were supposed to be. Raji Cells, A549s, U937s, I knew them all, worked with them every day, and never doubted that they were the cells that I thought that they were. I knew that some cell lines had been contaminated with the HeLa cell line. HeLa cells are very hardy and could spread quite easily from one flask to another. But I thought that was in the past. It turns out that there was more to the story than I realized. Cell lines have a provenance, like paintings or other works of art. They have an origin, a laboratory where the line was first isolated and propagated. From there, it may have been distributed to other laboratories and to repositories like the American Type Culture Collection or ATCC. Some cell lines are used by only a few laboratories, and some become used very widely and in a large number of biomedical disciplines. Whereas some paintings are intentionally forged, many cell lines have now been shown to be unintentionally forged. A recent article in the journal Science estimated that 20% of all immortalized cell lines are not what they were thought to be1.

Download original file2400 × 1999 px jpg View in browser You can attribute the author Show me how Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein targeted to the Golgi apparatus (orange), microtubules (green) and counterstained for DNA (cyan). Nikon RTS2000MP custom laser scanning microscope. National Institutes of Health (NIH).


Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein targeted to the Golgi apparatus (orange), microtubules (green) and counterstained for DNA (cyan). Nikon RTS2000MP custom laser scanning microscope. National Institutes of Health (NIH).

We now have better methods of identifying cell lines by their DNA, called short tandem repeat (STR) profiling, and scientific journals are beginning to require this testing for cell lines prior to publication. Currently, 28 scientific journals require STR profiling to establish cell line provenance prior to publishing a manuscript from a particular laboratory. Some scientists are also beginning to create catalogs of contaminated cell lines in an attempt to quantitate the damage done by some misidentified, but widely studied, cell lines. The same Science article, notes that the International Cell Line Authentication Committee (ICLAC) maintains a database of misidentified cell lines that now numbers 475 different lines. A cell line geneticist, Dr. Christopher Korch, recently estimated that just two of the immortalized cell lines that were found to be misidentified, HEp-2 and INT 407, have generated 5,789 and 1,336 articles in scientific journals, respectively. These studies cost an estimated $713 million dollars and generated an estimated $3.5 billion in subsequent work based on those papers1. This is because the usual trajectory for testing a new therapeutic modality, especially in cancer research, is to test a compound or technique in cell culture. It will then be tested in mice that express a tumor derived from the cultured cancer cells. If those studies are successful, the compound and/or technique undergoes further toxicity testing in other animal models before entering its first Phase I trial in human volunteers.

A lot of compounds that show early promise in cell culture and in cell line-injected mice turn out not to have efficacy in animal models or in human patients. Sometimes this is simply a matter of the compound being too toxic to organs or cell types that are not represented in the initial cell culture. Often, the reason why particular compounds or strategies fail is not known, and most granting agencies are not keen to fund laboratories to find out why things don’t work. I have wondered if the failure of some compounds or techniques is in part due to misidentified cell lines. I have also wondered if it is a reason why testing in animal models, particularly in animal models with spontaneously-occurring tumors, is necessary.

Testing anti-cancer compounds in models of spontaneously-occurring tumors in animals and/or testing in human tumor cells taken directly from patients and injected into mice (the ‘mouse hospital’ approach) is more time and resource intensive than screening in immortalized tumor cell lines. This approach, however, has the advantage of knowing that the investigator is not just treating misidentified HeLa cells in error. It is always necessary to go from in vitro cell culture models to in vivo animal models to confirm the viability of a therapy.

Science makes claim to no enduring wisdom, except of its method. Scientists only strive to be more right about something than we were yesterday, and efforts are underway to weed out misidentified cell lines. But the fundamental issues behind cell line misidentification highlight one of the reasons why we should not rely on immortalized cell lines without animal models, and why granting agencies should fund more studies to try to identify the disconnect between the results of in vitro and in vivo studies when things do not go as planned. That is a part of good science and part of creating better cell culture models to refine, reduce, and sometimes replace the use of animals in biomedical research.

Lisa Krugner-Higby, DVM, PhD

1) Line of Attack. Science. 2015. Vol. 347, pp. 938-940.

Mice Help Develop Molecular Imaging of Tumors

Can you follow the structural growth and metabolic activity of a developing tumor?   Such an advance would allow one to track how patients are responding to their therapies right away instead of having to wait weeks.  The video shows new research in the field of molecular imaging and yet another example of how the development of novel medical devices relies on the use of animals in research.

You can learn more here:

1: Mather SJ. Design of radiolabelled ligands for the imaging and treatment of cancer. Mol Biosyst. 2007 Jan;3(1):30-5. Epub 2006 Nov 14. Review. PubMed PMID:

2: Phelps ME. PET: the merging of biology and imaging into molecular imaging. J Nucl Med. 2000 Apr;41(4):661-81. Review. PubMed PMID: 10768568.

Regards

Dario

RNAi: Send in the Nanobots!

The publication of the preliminary results of a small clinical trial of a new therapy called RNA interference (RNAi) online in the scientific journal Nature is causing quite a stir in the scientific community this week.  A team led by Professor Mark E. Davis at Caltech targeted the delivery of a nanoparticle only 70 nanometers in diameter containing small interfering RNA (siRNA) to cancer cells in three patients with metastatic melanoma, which reduced the levels of a protein called RRM2 that is required for the tumour growth.  This trial is the result of over a decade of research in organisms as diverse as nematode worms, mice and monkeys, but why is the result of this trial so noteworthy? And what is RNAi anyway?

Cancer genes in human melanomas have been switched off. Image courtesy of the National Cancer Institute

If you have ever studied biology you will probably be familiar with the “central dogma of molecular biology”; it describes how our genes encode the proteins that are the building blocks, and indeed the builders, of all the cells in our bodies. The very short version is that our genes are made up of sequences of double stranded DNA consisting of the deoxyribonucleotides A,C, G and T, and these sequences are transcribed by a protein called RNA polymerase into matching sequences of the single stranded messenger RNA (mRNA) , made from the ribonucleotides A, C, G and U. Another protein complex known as the ribosome then translates the mRNA sequence into a corresponding sequence of amino acids that when completed make up a brand new protein.  Our new protein almost invariable undergoes further processing but we needn’t concern ourselves with that here.  RNAi is the process where an assembly of proteins named the  RNA-induced silencing complex (RISC) binds short double stranded segments of RNA that in turn target RISC to particular mRNA sequences to which they are complementary.  RISC breaks down the mRNA molecule, preventing production of its associated protein and effectively silencing the targeted gene. The beauty of RNAi is that it allows an organism to target specific mRNA molecules for destruction, and it is a mechanism for regulating the flow of genetic information whose importance we are still only beginning to appreciate.

RNAi was discovered only 12 years ago by Andrew Fire and Craig Mello through their basic research on the regulation of gene expression in the nematode worm Caenorhabditis elegans, a discovery which earned then the Nobel Prize in 2006. C.elegans is a popular model organism for scientists studying gene function and development, its small size and simple structure make it relatively easy to follow the fate of individual cells, while as an animal it shares many of its genes and biological processes with mammals.  This turned out to be the case with when in 2001 it was shown that RNAi helps mice to control hepatitis B infection, and scientists began to examine whether RNAi could be used therapeutically (1). To do this scientists made siRNA, an artificial version of the short double stranded segments of RNA that target RISC to complementary mRNA sequences, and early experiments in mice demonstrated that siRNA induced RNAi could reduce the levels of target proteins in mice.  The first human trials of RNAi began in 2004 for the treatment of wet age related macular degeneration and at first seemed promising, but suffered a setback when further research in mice revealed that the “naked” siRNA injected into the eye in these trials actually stimulated an immune response that was responsible for at least some of the benefits seen in earlier trials (2). This was a worry as an unwanted immune response might lead to an adverse reaction if the siRNA was injected into the bloodstream rather than a small part of the eye.

In recent years scientists have been developing technologies that allow injected siRNA to evade the immune system and target only those tissues where RNAi activity is desired,  reducing the quantity of siRNA that needs to be injected and also the risk of adverse  effects due to RNAi affecting off-target tissues. Mark E Davis, a professor of chemical engineering at Caltech and one of the scientists leading these efforts, uses polymers that assemble with siRNA to form a nanoparticle that resembles a tiny ball with siRNA at its centre.  The nanoparticle shell protects the siRNA from being broken down while it is circulating in the bloodstream, and then interacts with the cell membrane to help the siRNA enter a cell so that it can do its job.  Of course he didn’t want the nanoparticle to release its siRNA payload into any old cell so he attached a protein called transferrin as a targeting ligand to the nanoparticle. Tumour cells express far more of the transferrin receptor on their surfaces than normal cells, and the hope was that the nanoparticles would bind to tumour cells in preference to normal cells.  To test whether this would work Prof. Davis team injected the nanoparticles, containing a siRNA that targeted a cancer gene, into mice that had metastatic Ewing’s sarcoma(3). They observed that the transferrin labelled nanoparticle delivered the siRNA to the tumour cells, knocked down the activity of the target cancer gene and dramatically slowed tumour growth, and when the transferring ligand was removed his effect not seen.  They also observed that the nanoparticle did not stimulate the immune system or affect any of the major organs of the mouse, indicating that their method had solved safety problems seen in earlier RNAi trials.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion. Image courtesy of Derek Bartlett and the California Institute of Technology.

Prof. Davis and his colleagues next needed to identify an appropriate target for human trials of their nanoparticle siRNA delivery system, and decided to target the M2 subunit of Ribonuclease reductase (RRM2), a protein that is required for cell division and which has recently been the subject of a lot of research as a target for anti-cancer drugs.   They first used in vitro studies to identify a siRNA sequence that effectively targeted the RRM2 mRNA, which they named siR2B+5, and then demonstrated in mice that this siRNA could block the production of RRM2 and reduce the growth of tumours (4).  As a final safety evaluation prior to human trials they injected different doses of their nanoparticle  containing siR2B+5 and labelled with transferrin to cynomologus monkeys, whose RRM2 mRNA is targeted by siR2B+5 in exactly the same way as in humans,  and found that it was safe and did not produce any unwanted effect on the immune system (5).

The human clinical trial reported this week confirmed that transferrin-labelled nanoparticle injected into the bloodstream were safely delivered siR2B+5 to the tumours of metastatic melanoma patients, and that the siRNA knocked down the production of RRM2 protein by RNAi (6).  Of course this is only a preliminary result, at this stage we don’t know to what extent this experimental treatment will reduce tumour growth in these patients, let alone if it will cure their cancer. If it is a success it will probably need to be combined with other anti-cancer drugs to be fully effective, so it is good to know that thanks to animal research other nanotechnology based drugs such as Lipoplatin are in clinical trials that offer more potent anti-cancer activity with less toxicity than existing anti-cancer drugs. Nonetheless to focus on this uncertainty would be to miss why this small trial is causing such excitement; for the first time scientists have shown that it is possible to target RNAi therapy to a particular tissue type within the body, and that is a breakthrough that opens up a whole new area of medicine. The era of RNAi medicine has begun!

Paul Browne

1)      McCaffrey A.P., et al. “RNA interference in adult mice.” Nature Volume 418, pages 38–39 (2002) DOI: 10.1038/418038a

2)      Castnotto D. and Rossi J.R. “The promises and pitfalls of RNA-interference-based therapeutics” Nature Volume 457(7228), pages 426-433 (2009) DOI:10.1038/nature07758

3)      Hu-Lieskovan S. et al. “Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma.” Cancer Re. Volume 65 (19), Pages 8984-8992 (2005) DOI:10.1158/0008-5472.CAN-05-0565

4)      Heidel J. et al. “Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo” Clin. Cancer Res. Volume 13(7), Pages 2207-2215 (2007) DOI: 10.1158/1078-0432.CCR-06-2218

5)      Heidel J. et al. “Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.” Proc.Natl Acad. Sci. USA Volume 104(14), Pages 5715-5721 (2007) DOI: 10.1073/pnas.0701458104

6)      Davis M.E. “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles”  Nature Advance Online Publication 21 March 2010 DOI:10.1038/nature08956

From Mouse to Monkey to Humans: The Story of Rituximab

Modern advances in science have meant that our models of diseases have vastly improved. Be that in a dish in the laboratory, a computer simulation or through using a transgenic mouse, there have been developments across the biomedical field that have given us a greater understanding of diseases and how our bodies work.

This increase in knowledge has meant that we are finding may drugs already on the market can treat a variety of diseases – those involving the same pathway or cell type. This is precisely what happened this month with a drug called Rituximab.

Rituximab was licensed in 1997 for use in the treatment of Non-Hodgkin’s lymphoma (NHL) – a cancer where cells of the immune system called B-cells mutate and divide abnormally. The cancer then spreads around the body when the B-cells clone themselves in replication.

Since it’s initial approval for use in NHL, rituximab has been used to successfully treat advanced rheumatoid arthritis and has also been part of anti-rejection treatments for kidney transplants (both involve B cells. Then news came last week that it could even slow the progression of rheumatoid arthritis (RA) in the early stages of the disease.

Rheumatoid Arthritis

Rituximab is an interesting drug, as it is a chimeric antibody. This means that it contains portions of both human and mouse antibodies mixed together. The first papers reporting on rituximab were published in 1994. The first looked at its creation, and the second reported on the phase I clinical trials of the drug.

The human immune system works by using antibodies as their ‘messengers’. The antibodies contain multiple regions that allow them to work effectively. One part of the molecule binds with the foreign molecule; the other part then recruits the immune cells to destroy the molecule and eliminate it from the body.

The B-cells mutated in NHL and involved in RA are part of the human immune system and are responsible for making antibodies against ‘foreign invaders’. Mature forms of B-cells have a protein called CD20 on their surface.

The protein CD20 was the target for a team in San Diego (1) in 1994. Because NHL and RA are characterised by excessive levels of, or mutated B-cells, they looked at ways to reduce their numbers. The researchers determined that CD20 was the perfect target on the human B-cells as it was located on the surface of the cell and it didn’t mutate, move inside the cell or fall off in the life cycle of the B-cell. The team then produced an antibody that would attack CD20 itself, so it would bind to the outside of B-cells, flagging them to the immune system to be eliminated. They identified a mouse antibody that had high anti-CD20 activity.

They then constructed a “chimeric” antibody containing the variable domain of the mouse antibody, the portion that specifically binds CD20, along with the constant domain of human antibody, the portion that recruits other components of the immune system to the target.

The construction of a chimeric antibody (later named rituximab) was crucial, as the mouse antibody was unsuitable for direct use in humans. While the mouse antibody was able to bind to human CD20, it would not be able to then recruit the human complement system and immune cells that are needed destroy the “targeted” B cells. It would also quickly be recognised as foreign in the human body, and destroyed by the immune system, therefore by using a chimeric antibody with enough human characteristics, the antibody would not only recognise the human CD20 and target the immune system to it but would remain in the body long enough to destroy the B cells.

To test whether rituximab would work as hoped, they performed studies in cynomolgus monkeys. They choose this species because the constant domains of their antibodies are very similar to those in humans, unlike those of the mouse, allowing the chimeric antibody to function as it would in humans. Following administration of rituximab the number of B cells in the monkey’s bloodstream fell dramatically. The numbers were also reduced in the bone marrow (where B cells are produced) and the lymph nodes (where they are activated to target foreign molecules). Rituximab administration was non-toxic and in the weeks after treatment finished the number of B-cells slowly recovered. This is important as it demonstrates that the treatment didn’t harm the monkey’s bone marrow stem cells, an important consideration since these cells are required for a healthy immune system.

Rituximab was an ideal candidate to treat NHL and the promising results in monkeys prompted the scientists to conduct phase I clinical trials inhuman patients which confirmed that rituximab was safe and indicated that it could shrink tumors.

Evaluation of the effectiveness of rituximab involved many studies of patients with Non-Hodgkin’s lymphoma. While the initial clinical trial results varied, likely due to the differing sizes of tumors between the patients, they showed it was effective at reducing B-cell numbers and tumor size. Since it’s approval numerous clinical trials have confirmed that rituximab is an effective treatment for Non-Hodgkin’s lymphoma (3).

This month’s exciting study by Professor Paul-Peter Tak from the University of Amsterdam showed that rituximab in combination with the drug methotrexate could slow the progression of early stage rheumatoid arthritis (RA).

The study involved 755 patients diagnosed with RA within the last year. Methotrexate is already considered to be the best treatment for these patients and 12.5% of the patients taking only methotrexate in this study experienced significant reduction of their symptoms. However, compare this to the 30.5% of patients taking a combination of methotrexate and rituximab, and it is clear that rituximab is effective. Issues of cost have been raised in relation to rituximab, but if it turns out to be as effective in treating early RA as this study suggests, then it may ultimately save the health services and insurance companies money as slowing or stopping the progression of the disease will result in fewer patients needing the more expensive treatment and care required in advanced RA.

Emma Stokes

1) Reff M.E. et al. “Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20.” Blood Volume 83(2), Pages 435-445 (1994) PubMed: 7506951

2) Maloney D.G. et al. “Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma.” Blood Volume 84(8), Pages 2457-2466 (1994) Pubmed: 7522629

3) Schultz H. et al. “Chemotherapy plus Rituximab versus chemotherapy alone for B-cell non-Hodgkin’s lymphoma.” Cochrane Database of Systematic Reviews 2007, Issue 4. Art. No.: CD003805. DOI:10.1002/14651858.CD003805.pub2.

Can we protect the brain against tumor metastasis?

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.

Brain Metastasis From Lung Cancer

Brain Metastasis From Lung Cancer

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

Taking a BiTE out of non-Hodgkin’s lymphoma

Non-Hodgkin’s lymphoma (NHL) is a diverse family of cancers that affect a part of the body’s immune system known as the lymphatic system.  In NHL white blood cells become cancerous and develop into tumors at key points in the lymphatic system known as the lymph nodes, before spreading to other tissues.  About 50,000 Americans develop NHL every year, and while effective treatments such as Rituximab are available they don’t work for all patients and every year NHL kills nearly 20,000 people in the USA.

So it’s not surprising that the news that Blinatumomab, a novel treatment developed by the German firm Micromet, has performed very well in early clinical trials has been greeted with enthusiasm by cancer  research charities and the stock market alike.

In the trials (1) published this week in the prestigious journal Science, Blinatumomab was given to 38 NHL patients who had not responded to other treatments. In 7 of these patients tumors were found to have shrunk dramatically while in 4 patients the tumors disappeared completely.  Blinatumomab is the first BiTE antibody to enter clinical trials, and its innovative design combines a portion of an antibody, a protein produced by the immune system that binds to foreign material in the body, that targets the cancer cell with a portion of an antibody that binds to the T-cells of the immune system.  The BiTE antibody directs the T-cell to the cancer cell, which the T-cell then destroys.  Blinatumomab was developed after earlier studies using animal models of NHL had shown that antibodies could direct T-cells to target cancer cells, and it was hoped that the BiTE antibodies would do this more effectively. Of course before it was assessed in human clinical trials the BiTE antibody  Blinatumomab was studied in mouse models of NHL, since it was important to determine that they could target circulating immune cells to the tumors (2).

The contribution of animal research to the development of Blinatumomab was not limited to the evaluation of anti-cancer activity and pre-clinical safety, it was also crucial to manufacturing Blinatumomab itself*. BiTE antibodies are produced by heavily modifying a type of antibody known as a monoclonal antibody that binds very specifically to a particular target in the body. The first step of monoclonal antibody production is the immunization of an animal, usually a rodent, with the protein such as a cancer cell protein to which you wish the antibody to bind.  Animals are required for this step because an immune system is needed to produce the immune cells that recognize the target protein, and humans cannot be used for this process both because they cannot be injected with a disease-bearing agent in order to make antibodies, and because the human body does not produce antibodies to the human proteins that researchers often wish to target. Blood samples containing cells that produce antibodies against the foreign protein are then taken from the animal. These antibody producing cells are fused with a special cancer cell to produce a hybrid cell, or hybridoma, which can be grown almost indefinately in the petri dish and produce a large supply of monoclonal antibodies.  These monoclonal antibodies can then in their turn be modified to produce antibody derived drugs such as Rituximab and Blinatumomab.

We hope that larger trials of Blinatumomab against NHL confirm the results of this early trial, and that it will go on to be a valuable addition to the range of treatments available to fight this deadly disease.

* While hybridoma based monoclonal antibody production methods have been very successful, and are vital to current efforts to develop antibody based medicines, replacement technologies that require far fewer animals are currently being developed.  In the coming decades it is hoped that hybridoma based methods will increasingly be replaced by improving in vitro technologies, for example antibody phage libraries that display vast numbers of human or animal antibody fragments and can be used to identify antibodies specific for a particular target. This is a good example of the 3Rs in practise.

Paul Browne

1) Bargou R. et al. “Tumor regression in cancer patients by very low doses of a T cell-engaging antibody.” Science Volume 321(5891), pages 974-977 (2008).

2) Dreier T. et al. “T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct.” J. Immunol. Volume 170(8), pages 4397-4402 (2003)