Tag Archives: cancer

Research Roundup: Killing cancer cells, growing drugs in chicken eggs and more!

Welcome to this week’s Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • Growing drugs in chicken eggs may lower their cost. Interferon beta is a cell-signaling protein found in the body that acts against viruses, and is used to treat various illnesses ranging from multiple sclerosis to cancer. The downside is that the interferon protein molecule is extremely expensive to manufacture, costing between $300-$1000 for one microgram. Most dosages start at several micrograms; to treat multiple sclerosis, for example, the starting dose is 30 micrograms. Researchers developed a novel way to mass produce interferon beta using chickens genetically modified using CRISPR technology. While investigators still need to show that the chicken-produced protein is structurally the same as the protein in current medications, this technique could reduce the price of cancer drugs by at least 90%. Additionally, a drug produced using modified chickens, called Kanuma, has already been approved by the US Food and Drug Administration to treat Lysosomal Acid Lipase Deficiency. Researchers are currently writing up their results for publication.
  • How Studying Frog Embryos Is Helping Advance Tissue Engineering By Leaps And Bounds. The embryos and tadpoles of Xenopus frogs are transparent allowing researchers to observe their internal anatomy during development. This, and other features like their tolerance to extensive manipulation, make them easy to work with in a research setting. Frogs and humans have many similarities genetically and physiologically. Researchers at the University of Pittsburgh are working with frog embryos to understand the mechanical processes that guide the development of a complete living organism. They hope to use this to develop a tool that tissue engineers can use in regenerative medicine when building new tissue. Dr. Lance Davidson, professor at the University’s Swanson School of Engineering explains, “Many engineering fields have some kind of software or simulation tool that can take the guesswork out their designs before they actually start building. We are developing something similar for tissue engineers so they don’t have to rely on trial and error all the time.” They hope to apply this to support regenerative medicine therapies. Original source: Pitt’s Swanson School of Engineering

  • Engineered Proteins lower body weight in obese mice, rats, and primates. Obesity is an increasingly common problem throughout the world. Surgeries such as gastric bypass or sleeve are quite effective, however the procedure is highly invasive and can lead to permanent negative side effects. Because of these negative side effects, scientists are currently exploring what different types of proteins our bodies secrete during metabolism. One promising protein that they identified was growth differentiation factor 15 (GDF15). By treating obese mice with GDF15, scientists discovered that mice reduced how much food they were eating leading to a reduction in body weight, and had healthier metabolism. They then tested this treatment in obese rats and cynomolgus monkeys, and found the same results. Through more intensive tests they also discovered that treatment with GDF15 delays gastric emptying, changed food preferences, and activated areas of the gut-brain axis. This work is a great example of scientific discoveries following the path of mouse to rat to non-human primate and, hopefully one day soon, human. This research was published this week in Science Translational Medicine.
  • Zebrafish research guides new therapy possibilities for rare genetic disorder. Alagille Syndrome is a rare (1 in 100,000 births), potentially life-threatening genetic disorder that affects the heart, liver, and kidneys among other body systems. New research using zebrafish has helped to identify the tissues and genes which are important to the development of liver duct cells, and how the mutation associated with Alagille Syndrome causes development to go awry. The team, based out of Sanford Burnham Prebys Medical Discovery Institute, hopes that this discovery will aid in the development of regenerative therapies that will restore liver function, and possibly prevent the need for liver transplant in certain patients with this disorder. This research was published in Nature Communications.

Zebrafish: Wellcome Trust Sanger Institute

  • New compound targets energy generation killing cancer cells. Sperm cells can generate energy and they can do so in harsh conditions because they strategically contain mitochondria in their “head”. Cancer cells, can also survive under harsh conditions, and they can adapt to a shortage of nutrients by reprogramming the energy generation system. Cancer cells, in contrast to normal cells, contain an enzyme called FerT — and unsurprisingly — the only other cell containing this enzyme is sperm. Researchers hypothesized that by disrupting the activity of FerT in cancer cells – they would starve cancer cells of energy and that they would die. To this end – they created a synthetic orally administered compound (E260), and found in mouse cancer model – that indeed, cancer cells are killed. They also check other normal cells and found them to be unaffected. This research was published in the journal Nature Communications.

Research Roundup: Snail venom and cancer, reversal of advanced heart failure and more!

Welcome to this week’s Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • Secrets found in snail venom may help treat cancer. Cone snails are marine mollusks that are found worldwide in warm climates. Usually reclusive, cone snails will produce a venomous sting when threatened using a single, harpoon-like tooth. They also use this venom to immobilize their prey, which are often much bigger and faster than the snails themselves. By examining the molecular makeup of cone snail venom, researchers are learning how a single toxin, which typically only affects the central nervous system, can also impact the immune system. This information may help develop therapies for cancers that involve uncontrolled overproduction of certain cells, such as gastric, breast, and lung cancers. Published in Scientific Reports.

In the wild, cone snails harpoon their prey as it swims by. In the lab, the cone snail has learned to exchange venom for dinner. Here, a snail extends its proboscis and discharges a shot of venom into a latex-topped tube.
Credit: Alex Holt/NIST

  • Scientists reverse advanced heart failure. Heart failure is one the most common reasons for hospital admittance in individuals 65 years or older. It occurs when the heart is unable to pump blood sufficiently to maintain the body’s needs. This week, researchers were able to reverse severe heart failure in a mouse model, by silencing the Hippo pathway. The Hippo pathway is associated with cell death, which occurs, for example, when heart tissue is starved of oxygen. Dr. James Martin, the corresponding author on this study states “Heart failure remains the leading cause of mortality from heart disease. The best current treatment for this condition is implantation of a ventricular assist device or a heart transplant, but the number of hearts available for transplant is limited”. This mouse model, which mimics the human condition of advanced heart failure, is therefore an exciting new avenue for further investigation into measures which limit the debilitating consequences of heart failure. This study appears in the journal Nature.
  • Zebrafish recover faster from stressful situations when housed together. Zebrafish are a small, schooling minnow-like species increasingly used in many aspects of biomedical research. A new study shows that when zebrafish are housed together after a stressful procedure, they recover faster, resume normal behaviours and even have lower levels of stress hormone than fish housed alone. The study also demonstrated that stress hormone levels can be measured non-invasively by sampling the water directly from fish tanks. Refining how we work with zebrafish, and discovering better ways to provide for their welfare needs are important aspects of doing valuable life-saving research with these animals. This research was published in the journal Animal Behavior.

Zebrafish: Wellcome Trust Sanger Institute

  • The link between caesarean sections, the microbiome, and obesity. Caesarean section, a.k.a. C-section, is a life-saving practice for delivering 10-15% of human newborns. However, C-section is also overused in the developed world with some regions delivering 43% of newborns by C-section. Although this practice is quite common, scientists and medical doctors understand little about the long-term effects of C-section. This week, scientists have uncovered evidence that being delivered by C-section is linked to an increased risk of obesity in mice. This link between C-section and obesity deals with the gut microbiome. When humans, or laboratory mice, are delivered normally they travel through the vaginal canal and get exposed to vaginal microbiota. C-section circumvents the vaginal canal and thus the newborns do not get exposed to this vaginal microbiota. Research published this week in Science Advances indicates that mouse pups born by C-section weigh significantly more than those born normally. They also have a different gut microbiomes. This research does not necessarily mean humans born by C-sections are at higher risk for obesity, because human newborns often get antibiotics immediately after delivery and mice are fostered to new mothers after being delivered by C-section. Nonetheless, this is a great step towards further understanding the consequences of C-section deliveries.
  • The validity of studies on the transplantation of tumours to mice questioned. This week ,a study published in the journal Nature Genetics, described changes in the genome of tumor tissue implanted into immunodeficient mice that may affect interpretation of research results. Human tumors can be studied in cell culture medium or by implanting cultured cells into immunodeficient rodent models. However, the process of ‘immortalization’ of cells grown in artificial culture medium alters the cells in ways that limit their usefulness as a model in tumor biology.  As an alternative, tumors collected from patients can be implanted directly into rodents (PDX or patient-derived xenograft avatars) to study their activity and response to therapeutic drugs.  This approach has been thought to better replicate the behavior of tumors in human patients with improved predictability of the model as a desired outcome. However, the study in Nature Genetics by Uri Ben-David and colleagues found that the unstable genome in many tumors continues to change after implantation into the mouse, and can accumulate mutations that differ in behavior and response to chemotherapeutics from the original patient tumor. These findings do not negate the value of the PDX avatar model, but do highlight the need for further investigation to determine how the genomic changes that occur affect the interpretation of results derived using this type of model.

Of Mice and Mammaries, Part 1: There’s something in the milk

In light of Breast Cancer Awareness Month, Justin Varholick traces how mice have helped breast cancer research over the past century. In the first post this 4-part series, we look at advances made from 1854 to 1940, including the understanding of the role of breast milk in causing certain types of tumors. 

Image credit: Jackson Labs

Breast cancer is one of the most serious forms of cancer facing women. Each year, over 300,000 women in the U.S. will be diagnosed with breast cancer, and it is estimated that 40,610 women will die from it in 2017 alone. Thankfully, death rates from breast cancer have dropped almost 40% from 1989 to 2015, and there are over 3.1 million breast cancer survivors living in the U.S. today.

Breast cancer grows and spreads through many stages, and can start in different parts of the breast. Some types of breast cancer cause lumps, others form no lumps. Some forms of it spread very quickly throughout the body, while others spread more slowly. Because breast cancer spreads and forms at different rates and in different areas of the breast, treating it is no easy task. It is also unlikely that we will one day have a “cure” for breast cancer — one size cannot fit all.

Despite the complicated nature of breast cancer, scientists feel a responsibility to understand it as much as possible in efforts to find new treatment methods and forms of a cure. Over the years they have made great strides in their research by studying mice. These mice serve as an essential step between early research on mammary cells and clinical trials in humans.

Over the course of this month, I will highlight some of the key findings scientists have discovered about breast cancer through their studies in mice.

1854 to 1903 — The first mouse mammary tumors

The first discovery of a mammary tumor in a mouse was in 1854. In these early days, scientists were able to find tumors spontaneously growing in female mice kept as pets and in the wild. Although they were able to detect and describe these tumors, it was difficult to understand where they came from, and how they grew and possibly spread or metastasized.

Thankfully in 1903, Dr. Carl Jensen developed a line of “high tumor” mice that readily grew mammary tumors, which could be easily transplanted to other mice. By transplanting the tumors in other mice, they could measure how and where the tumors spread, in otherwise healthy mice.

During this time in history, 1.2* women per 1,000 died from breast cancer in the U.S. Today it is around 0.13 women per 1,000. (*at this time we only had reports on the number of white women in the U.S.).

1933 to 1940 — There’s something in the milk

After bringing mice into the laboratory and thoroughly studying their biology, a great discovery was made — there was something in the milk. This discovery was made by Scientists at Jackson Laboratories, in Bar Harbor, Maine. They bred “high tumor” mice with “low tumor”* mice and found that offspring were more likely to get mammary tumors if they had a “high tumor” mother. Although some scientists were able to replicate this finding in other labs, very few were convinced there was something in the milk — they believed it was passed down through the genes. (* an extremely low number of “low tumor” mice were found with mammary tumors; because of this scientists could not call them “no tumor” mice.)

To answer whether there was either something in the genes or the milk, Dr. John J. Bittner did a more complex study 3 years later. In this key study, Bittner cross-fostered mouse pups from “high tumor” and “low tumor” mice to opposite mothers (see diagram). This method allowed him to determine whether the parent’s genes or the foster mother’s milk lead to mammary tumors. If it was the genes then “high tumor” offspring would have tumors whether they had “high tumor” or “low tumor” foster parents. If it was the milk then any offspring nursed by “high tumor” mothers would get tumors.

Through this experiment, Bittner found out that milk was a key factor. “Low tumor” pups cross-fostered to “high tumor” mothers had many mammary tumors, while “high tumor” pups cross-fostered to “low tumor” mothers had very few tumors. “High tumor” pups nursed by their own mothers, however, had the highest rates of tumor growth. It didn’t always matter who the parents were, it also mattered who nursed the pups. This verified that indeed there was something in the milk. This something was labeled as the Mouse Mammary Tumor Virus (MMTV).

Dr. Bittner was often heard stating that he only studied the milk because nobody else wanted it — they all wanted to study the genes.

To be continued…

Tune in next week to read what we learned about the milk virus, MMTV, and what we did with this new power!

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).
  2. Holen I, Speirs V, Morrissey B, Blyth K. (2017). In vivo models in breast cancer research: progress, challenges and future directions. Dis Model Mech. 10(4).
  3. Tarone RE, Chu KC. (1992). Implications of birth cohort patterns in interpreting trends in breast cancer rates. J Natl Cancer Inst. 84(18).

Does talcum powder cause ovarian cancer? Weighing up the human and animal studies

In this article, Justin Varholick, investigates the evidence on whether talcum powder can cause ovarian cancer. Over the years, several courts have ruled that talcum powder can cause ovarian cancer, while the scientific evidence suggests otherwise. In light of Ovarian Cancer Month, it is important to highlight how animal and human studies can improve our understanding of the disease, and prevent misinformation spread from the media. This article outlines that both animal and human studies are not perfect. Animal studies sometimes do not have proper controls and human studies suffer from bias. The current research suggests no direct link between talc and cancer, but more research is certainly necessary.

Ovarian cancer is a serious disease affecting around 22,000 women in the United States and contributing to around 14,000 deaths each year. Since the 1960s the American public has questioned whether the use of talcum powder – for soothing dry skin, absorbing sweat, and preventing chafing of the thighs — increases women’s’ risk for ovarian cancer. This speculation began after acknowledging the risks of asbestos and public theories that asbestos was in talc products; however, cosmetic grade talc undergoes strict quality control and does not contain asbestos.

Image by Austin Kirk

Multiple studies on rodents, non-human primates, and humans have investigated the link between talc and cancer since the 1960s. Overall the results are inconsistent; some studies suggest talc is associated with ovarian cancer while others suggest talc is not carcinogenic. Recently, despite these inconsistencies, a Los Angeles jury ordered Johnson & Johnson to pay $417 million to a woman who blamed her terminal ovarian cancer on the use of baby powder — this is just one of many lawsuits against Johnson & Johnson over their talc powder. In light of this recent event I would like to delve into the animal and human studies investigating the link between talcum powder and ovarian cancer.

Is talcum powder a carcinogen?

Empirical studies first began on rodents such as hamsters, rats, and mice; however, these studies only focused on whether talc was a carcinogen in general. Researchers chose rodents because it is relatively easy to systematically administer talc to rodents via inhalation. Furthermore, rodents — especially the laboratory rat — are particularly sensitive to forming malignant tumors in the lungs when exposed to chemicals via inhalation regardless of the chemical itself. Therefore, by using rodents there is an increased chance of detecting an effect of cancer following exposure to talc via inhalation — if one is present. It is important to note here that although humans are exposed to talc by inhalation or via topical application, the specific method of applying talc is not important when determining general carcinogenicity.

For one of the first studies investigating talc exposure and cancer in rodents, researchers first gathered information on how much baby powder human infants were regularly exposed to – although infants are usually exposed via topical application and rodents are exposed via inhalation. Using this information they designed an experiment using hamsters and exceeded the amount of talc human infants are normally exposed to by 30 to 1700 times — depending on the experimental treatment group. The scientists also formed a control group that was exposed to a negative dust control; titanium dioxide. This control is important because increased levels of dust in the air can lead to chronic inflammation of the lungs, which increases the risk of malignant tumors — independent of particle type (e.g. talc powder, titanium dioxide, toner, carbon black, etc.). Controlling for dust and exceeding levels of normal exposure, the study reported no difference between the groups in body weight, survival, or signs of cancer in the larynx, trachea, lungs, liver, kidney, stomach, uterus, ovaries, or testes of these hamsters.

Further studies were conducted on rodents — specifically mice and rats — that did find an effect linking cancer to talc; however, these studies were confounded. One study in particular found that female rats and mice exposed to high levels of talc via inhalation for 4 months had a higher risk of lung cancer. Unfortunately, this study did not use a titanium dioxide control group, thus the finding could be an artefact of chronic inflammation from air particles — as discussed above. Furthermore, this study was unable to identify another biological mechanism beyond chronic inflammation responsible for the onset of cancer.

In summary, these rodent studies allowed scientists to exceed normal exposure levels and use an animal with increased sensitivity to the treatment in question. However, proper control groups must be used to help elucidate whether the effect is an artefact. Importantly, these studies were only interested in whether talc is a possible carcinogen, not whether ovaries exposed to talc have increased risk of cancer specifically. Overall, these studies were unable to find a link between talc and risk of cancer, beyond chronic inflammation from increased levels of air particulates.

Can talcum powder be found in the ovaries?

Some studies in animals and humans have been particularly focused on finding a link between talc use and ovarian cancer — not just whether talc is a carcinogen. To understand the plausibility of this link, these studies first needed to establish whether it is possible for particles of talc to migrate into the genital tract after being applied topically to the perineal region (area between vagina/scrotum and anus). A simple understanding of biophysics led many to conclude that it was impossible for the particles to travel up the vagina, cross the cervix, travel through the uterus, and then “swim” upstream through the oviducts; without being assisted by some form of locomotion. Nonetheless, some studies using animals investigated whether it was a possibility. Specifically, one study using female cynomolgus monkeys (Macaca fascicularis) — an animal model anatomically and physiologically comparable to human female — investigated whether carbon black particles could reach the oviducts or ovaries. This study was unable to conclude that carbon black particles could indeed travel up to the oviducts or ovaries.

Image by Noveprim

Further studies were done with human females that applied talcum powder to their underwear or perineal region daily that also had ovarian or pelvic cancer; which required surgical removal of the ovaries.  After removing the ovaries, scientists used microscopy techniques to scan the ovaries and identified low numbers of particles that were relatively small in size in about 50–75% of cases (multiple studies). Thus, although talc can be found in or around ovarian tissue the amount found was considered too small to cause ovarian cancer. It has also been noted that findings from these studies were widely inconsistent and were confounded by women lying in a supine or Trendelenburg position — which may aid in the surgery of the pelvic region but is also used to aid in vitro fertilization.

Thus, studies in both animals and humans cannot definitively suggest talc can translocate from the perineal region to the ovaries, which may be necessary for the talc to affect the ovaries. Nonetheless, both animal and human studies have been limiting; studies with monkeys only used a particle similar to talc and human studies involved a lying position that aided in the migration of talc up the genital tract.

How many women using talcum powder get ovarian cancer?

Two types of human studies have investigated, and continue to investigate, the link between talc and ovarian cancer; case-control and cohort studies. The case-control studies gather a group of women diagnosed with ovarian cancer and a group of women with no ovarian cancer. They then ask all women to retrospectively discuss their use of talc on the genital area throughout their life — noting frequency and average amount. The obvious downside to this type of study is that it is open to reporting bias. Some women may forget when or how often they used talc, while others may overestimate their use and further bias may occur if there is an expectancy that talc may have contributed to the onset of ovarian cancer. In contrast, the cohort studies gather a group of women early in life and then have them report in real-time throughout their life how often they use multiple products — including products with talc. After several decades they then compare how many women are diagnosed with ovarian cancer and used talc products, diagnosed with ovarian cancer and did not use talc products, etc. Cohort studies, however, are often limiting because few women are actually diagnosed with ovarian cancer compared to those that are not.

A recent meta-analysis, published this year, gathered 24 case-control and 3 cohort studies investigating the use of talc on the perineal region and its relation to ovarian cancer. Gathering all of these studies into a single analysis, they found that talc powder use on the perineal region is associated with a small increased risk of developing ovarian cancer; however, case-control studies largely contributed to this association — which have obvious disadvantages as outlined above. This positive association was also limited to a single type of ovarian cancer; identified as serous carcinoma — the most common type of ovarian cancer (types of ovarian cancer). Importantly, if reporting bias is affecting the case-control studies, then the association between talc use and ovarian cancer should not be limited to a single type of ovarian cancer. The authors also note that publication bias may also be affecting the case-control studies, meaning that some hospitals may gather information about talc use and ovarian cancer but do not publish their findings because they do not find a link between the two.

In summary, studies with humans do suggest that there is a small positive association between talc use and ovarian cancer; however, these studies are largely limited to case-control studies which have disadvantages of reporting and publication biases. Furthermore, these studies can only tell us about the relative risk of ovarian cancer when using talc. They cannot tell us about the biological basis linking talcum powder use to cancer.

Talcum powder does not cause ovarian cancer

The current evidence from both animal and human studies does not suggest that talc can be directly linked to ovarian cancer. However, both animal and human studies are not perfect. Studies using animals sometimes lack important controls and are not able to properly investigate the specific question at hand without proper animal models (i.e. cynomolgus monkeys). However, animals can be utilized in investigating whether talc is a carcinogen in general because some are especially sensitive to different types of treatments. Studies with humans also have disadvantages due to limitations of subject pools and biases. Despite this, studies with humans somewhat consistently find a link between talc and ovarian cancer, thus humans may be particularly sensitive to talc beyond other animals — although this is highly unlikely given that studies on other mammals suggest no direct relationship.

Importantly, there are many more studies on animals and humans that investigate the link between talc and cancer that I did not include in this brief discussion. Therefore, it is important to note that in a recent review in 2015, the Cosmetic Ingredient Review Expert Panel reported that talc is safe to use in standard practices with normal concentrations. They also note that there is:

  • Absence of persuasive evidence that talc can migrate from the perineum to the ovaries
  • Lack of consistent statistically significant positive associations across studies
  • Failure to rule out plausible alternative explanations of statistically significant results, including biases, risk factors, and exposure to misclassifications
  • Absence of a plausible biological mechanism
  • Lack of credible, defensible evidence of carcinogenicity from results of epidemiological studies of occupational exposures and animal bioassays

Thus, more research is necessary to determine whether talc is linked to ovarian cancer, despite what the Los Angeles courts might say.

Justin Varholick

 

References

Berge, W., Mundt, K., Luu, H. and Boffetta, P. 2017. Genital use of talc and risk of ovarian cancer: a meta-analysis. European Journal of Cancer Prevention.

Fiume, M.M., Boyer, I., Bergfeld, W.F., Belsito, D.V., Hill, R.A., Klaassen, C.D., Liebler, D.C., Marks, J.G., Shank, R.C., Slaga, T.J., Snyder, P.W. and Andersen, F.A. 2015. Safety assessment of talc as used in cosmetics. International journal of toxicology 34(1 Suppl), p. 66S–129S.

Reid, B.M., Permuth, J.B. and Sellers, T.A. 2017. Epidemiology of ovarian cancer: a review. Cancer biology & medicine 14(1), pp. 9–32.

Wehner, A.P. 2002. Cosmetic talc should not be listed as a carcinogen: comments on NTP’s deliberations to list talc as a carcinogen. Regulatory Toxicology and Pharmacology 36(1), pp. 40–50.

Research Roundup: Biosensors, breast cancer and the benefits of antiretrovirals

Welcome to this week’s (slightly late!) Research Roundup. These posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s Research Roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • A new experimental technology can monitor and maintain drug levels in body. The device has a biosensor to monitor drug levels in the body; this can relay information every few seconds to a control unit and pump, which releases additional drugs as necessary. Using rabbits, the researchers were able to keep a constant dosage among all animals in their study – despite physiological and metabolic differences between individual animals. Taking it a step further, the research team introduced secondary drugs that, due to acute drug-drug interactions, would disrupt the levels of the initial drug. However they found levels of the initial drug were stabilised by the sensor.  This paper was published in Nature Biomedical Engineering.

Image courtesy of the Soh Lab, Stanford.

  • A gene associated with the growth of cancer cells is also implicated with the growth of stem cells. Previous research by this group has implicated the high-mobility group (HMG) gene in the formation of polyps, abnormal growths projecting from the intestinal lining that can be precursors of cancer, in mice. Examining the intestinal cells of these mice localized the HMG active gene and its protein to stem cells buried within the deep grooves in the intestinal lining. These stem cells carrying the HMG gene multiplied far more rapidly and also increased the number of Paneth cells, a type of niche cell known to support intestinal stem cells. This research provides an exciting avenue for future research into processes that could disrupt cancer growth and prevent tumour progression. This study was published in Nature Communications.

  • Young people who contract HIV in the UK can now expect to live to a near-normal age thanks to anti-retrovirals. A study in the Lancet of almost 90,000 people showed, “Patients who started Anti-Retroviral Therapy (ART) during 2008–10 whose CD4 counts exceeded 350 cells per μL 1 year after ART initiation have estimated life expectancy approaching that of the general population”. This is 10 years longer than those who started ART in 1996. This breakthrough owes much of its success to animal research that eventually lead to such clinical trials in humans. For example, the ability of AZT, an anti-retroviral medicine more commonly known as Retrovir and Retrovis, to act against HIV (without toxic side effects) was discovered in mice and rats.

How zebrafish help advance cancer research

Do sharks get cancer?

Despite the widely touted myth that sharks do not develop cancer, fish of all species do occasionally develop spontaneous tumours. This is of course also true for the most common of laboratory fish, the zebrafish. In this article, I will give you a brief overview of how the unique properties of the zebrafish have been exploited by scientists to generate very useful models to study the molecular basis of various cancers.

The use of zebrafish in cancer biology goes right back to when scientists first started using them in the lab, at which point it was noticed that they spontaneously develop various kinds of tumours. However, using these naturally occurring malignancies to study cancer development is rather impractical – not only would you need a lot of fish due to the rarity of these cancers, but there would also be a lot of heterogeneity as to what kinds of tumours develop. This is clearly not ideal if you want to study the molecular basis and treatment options of one particular cancer.

From disease to model

Subsequently, carcinogenic chemicals were used to speed up the onset of cancer development. However, similar to using naturally occurring tumours, this strategy is not terribly useful for studying one particular kind of cancer, as the resulting tumours can still be very diverse(although some substances tend to always cause the same type). This approach is mostly used to identify cancer-causing chemicals during human and environmental safety testing.

To study one specific cancer type in detail, scientists started to create zebrafish carrying particular loss of function mutations (i.e. genes that lose activity due to a change), or overexpressing certain cancer-causing oncogenes (i.e. genes that cause cancer when they are overly active). Usually, this leads to the early development of only one – or at most a few – types of cancer. The first of these more specific models were acute lymphoblastic leukaemia (ALL) models, but nowadays there are models for cancers of various tissues, ranging from the brain to the pancreas.

Most of these mutant models were originally created using mutagenizing drugs followed by screening for a phenotype, but recently the research community has shifted to more targeted techniques. These make use of novel genome editing tools, such as the CRISPR-Cas9 system to switch off certain genes. The overexpression of specific genes on the other hand was usually achieved using proteins called transposases to integrate novel genetic information, but very recently the CRISPR-Cas9 system has also been tweaked to do the same.

Why study cancer in fish?

So why would anyone bother to go through this effort and do all this in fish, if we can just use the more closely related mice or rats? Apart from the lower expense and easier generation of large numbers of fish, the main reason why fish are used is that visualizing particular cells is much easier than in other organisms. This is mainly due to two factors: the existence of various transgenic fish lines in which a particular cell type is labelled, and the existence of transparent adult fish (the casper fish, as below).

Casper_fish

Transparent fish like these Casper fish shown here allow researchers to track cells inside the body of adult fish much more easily than ever before

The ease of labelling specific cell types has been exploited elegantly for studying the clonal expansion of cancer cells that drives tumor growth in vivo as it happens, as well for the study of cancer metastases. Now that adult transparent zebrafish have enabled even easier in vivo imaging, the approach has been used successfully to visualize the process by which metastases arise and cancer cells distribute throughout the body.

Understanding the origins of melanoma

A recent paper from Charles Kaufman of the Harvard Stem Cell Institute and colleagues nicely illustrates how these advantages can be very powerful indeed. In this paper published in Science magazine, the researchers used a zebrafish melanoma model that they had developed a few years earlier which expresses gene variants associated with the cancer in humans, and combined this with a newly developed transgenic zebrafish line, in which cells expressing a gene known as Crestin, which is involved in early neural development , are labelled in green. The Crestin gene is normally not expressed in adult humans, but is switched on again in melanomas. This is also why this combination is interesting; emerging melanoma cells will re-express the normally silent gene and be labelled fluorescently.

This method allowed the researchers to track melanoma development from the very first tumour cell to the macroscopically visible tumour comprised of millions of cells. The very early changes that have to occur for cancer to develop can now be studied at much greater detail than before, as these very early tumorigenic cells are extremely hard (or completely impossible) to distinguish from normal cells if they are not labelled. In this specific case the researchers identified the activation of several gene pathways that are usually involved in neural crest development in the embryo as key events in the initiation of melanoma, and believe that their findings could lead to a new genetic test for suspicious moles in patients. Their work suggests a model of cancer development where normal tissue becomes primed for cancer when oncogenes are activated and tumour suppressor genes are silenced or lost, but where cancer develops only when a cell in the tissue reverts to a more primitive, embryonic state and starts dividing.

This paper increased our understanding of the underlying biology of the very early stages of tumour development, and a detailed understanding of these early steps might be very important when developing preventative or therapeutic drugs.

Image: Kaufman, C.K., et al, 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

Image: Kaufman, C.K., et al, 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

In summary, the field of zebrafish cancer biology has made great advances in the last decade and will continue to do so with the increasing popularity of genome editing techniques. The easy visualization of particular cell types leads to distinct advantages of using zebrafish, particularly for the study of metastases and the very early stages of cancer development.

Jan Botthof

References
Kaufman, C.K., Mosimann, C., Fan, Z.P., Yang, S., Thomas, A.J., Ablain, J., Tan, J.L., Fogley, R.D., van Rooijen, E., Hagedorn, E.J. and Ciarlo, C., 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

Lung cancer immunotherapy, from PD-1 knockout mice to clinical trials

This morning many news outlets, including the BBC, covered a very promising development in lung cancer therapy; the successful clinical trial of the cancer immunotherapy Nivolumab in 582 patients with advanced lung cancer. While the extension of survival was modest in most patients, it is to be remembered that these were patients with advanced lung cancer, which is notoriously difficult to treat, so to see the survival time doubling in some patients was quite dramatic. Future trials will examine whether greater benefits are seen when Nivolumab is given earlier in the course of the disease.

Dr Alan Worsley, Cancer Research UK’s senior science information officer, told the BBC that harnessing the immune system would be an “essential part” of cancer treatment, and adding:

This trial shows that blocking lung cancer’s ability to hide from immune cells may be better than current chemotherapy treatments. “Advances like these are giving real hope for lung cancer patients, who have until now had very few options.”

Nivolumab works by blocking the activation of the PD-1 receptor protein found on the surface of many of the immune cells that infiltrate tumours. Another protein named PD-L1 binds to PD-1 and initiates a regulatory pathway that leads to the immune response being dampened down. Usually this is a good thing as it maintains immune tolerance to self-antigens and prevents auto-immune damage to healthy tissue, but unfortunately many solid tumour cells, such as lung cancer cells, also secrete PD-L1, and by activating PD-1 can evade destruction by the immune system. By blocking PD-1 Nivolumab turns off this protective mechanism and allows the immune cells to detect and destroy the tumour cells.

X-ray of a lung cancer patient. Image credit:

X-ray of a lung cancer patient. Image credit: “LungCACXR” by James Heilman, MD – Own work.

So how was this discovered? This is where the knockout mice come in. Scientists had observed in the 1990’s that PD-1 was highly expressed on the surface of circulating T- and B- immune cells in mice, but didn’t know what role PD-1 played, suspecting that it may be involved in increasing the magnitude of the immune response. To examine the role of PD-1 researchers at Kyoto University in Japan led by Professor Tasuku Honjo created a knock-out mouse line where the PD-1 gene was absent, and observed that this lead to some immune responses being augmented. In a paper published in 1998 they reported than rather than being an activator of the immune response PD-1 was actually involved in dampening down the immune response (1).

Subsequent studies in a range of PD-1 knockout mouse strains over the next decade explored the role of PD-1 in regulating the immune system, and also demonstrated that its ligand, PD-L1, could block immune-mediated tissue damage (2).  At the same time as these studies were taking place other research was demonstrating that PD-L1 was produced at high levels by tumour cells, first in   renal cell carcinoma in 2004 (3), but later in many other solid tumours including in lung cancer (4), and that this expression was associated with a decrease in the immune response to the tumour and a poorer prognosis.

This raised an obvious question: would blocking PD-1 improve the immune response against these tumours?

Work was already underway to find out. A paper published in 2007 by scientists from Nara Medical University in Japan demonstrated that blocking PD-L1 binding to PD-1 with monoclonal antibodies enhanced the immune response against established tumours in a mouse model of pancreatic cancer and acted synergistically with chemotherapy to clear the tumours without obvious toxicity (5). Subsequent studies with other monoclonal antibodies in a range of mouse and in vitro models of cancer showed similar results, including the humanized monoclonal antibody MDX-1106, now called Nivolumab, which was obtained by immunizing mice which had been genetically modified to produce human antibodies with human PD-1 (6).

Laboratory Mice are the most common species used in research

Cancer Immunotherapy – adding another accomplishment to an already impressive CV!

MDX-1106/Nivolumab showed promising results in a phase 1 trial against metastatic melanoma, colorectal cancer, castrate-resistant prostate cancer, non-small-cell lung cancer, and renal cell carcinoma, and following larger clinical trials (7) it was approved by the FDA for the treatment of melanoma that cannot be removed by surgery or is metastatic and no longer responding to other drugs, and more recently for metastatic squamous non-small cell lung cancer.

The story of the development of anti-PD-1 cancer immunotherapy is an illustration of how basic or fundamental biological research in animals informs medical science, and drives the discovery of new therapies. As cancer immunotherapy begins to transform the treatment of many previously untreatable cancers, it is well worth remembering that this revolution has its origin in the hard work of countless scientists working around the world, many of whom could only have guessed at the time where their efforts would eventually lead.

Breaking news, 1 June 2015: In another exciting report from the American Society of Clinical Oncology meeting in Chicago, researchers have reported that in a clinical trial of 945 patients with advanced metastatic melanoma a combination of Nivolumab with  Ipilimumab (another cancer immunotherapy that works through a different mechanism) stopped cancer advancing for nearly a year in 58% of cases, with the cancer still stopped in its tracks in many patients when the study period had ended. This is substantially greater effect than is seen with existing therapies, including Ipilimumab when administered alone, and shows how powerful cancer immunotherapies may be when two or more are combined.

Paul Browne

References:

  1. Nishimura H1, Minato N, Nakano T, Honjo T. “Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses.” Int Immunol. 1998 Oct;10(10):1563-72. PubMed: 9796923
  2. Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH, Lichtman AH. “Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart.” Circulation. 2007 Oct 30;116(18):2062-71. PubMed 17938288
  3. Thompson RH1, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, Krejci KG, Lobo JR, Sengupta S, Chen L, Zincke H, Blute ML, Strome SE, Leibovich BC, Kwon ED. “Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.” Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17174-9. PubMed:15569934
  4. Zhang Y1, Huang S, Gong D, Qin Y, Shen Q. “Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer.” Cell Mol Immunol. 2010 Sep;7(5):389-95. doi: 10.1038/cmi.2010.28. PubMed: 20514052
  5. Nomi T1, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, Nakamura S, Enomoto K, Yagita H, Azuma M, Nakajima Y. “Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.” Clin Cancer Res. 2007 Apr 1;13(7):2151-7. PubMed:17404099
  6. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, Stankevich E, Pons A, Salay TM, McMiller TL, Gilson MM, Wang C, Selby M, Taube JM, Anders R, Chen L, Korman AJ, Pardoll DM, Lowy I, Topalian SL. “Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.” J Clin Oncol. 2010 Jul 1;28(19):3167-75. doi:10.1200/JCO.2009.26.7609. PubMed: 20516446
  7. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, Leming PD, Lipson EJ, Puzanov I, Smith DC, Taube JM, Wigginton JM, Kollia GD, Gupta A, Pardoll DM, Sosman JA, Hodi FS. “Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab.” J Clin Oncol. 2014 Apr 1;32(10):1020-30. doi: 10.1200/JCO.2013.53.0105. PubMed:24590637