Autism Spectrum Disorder (ASD) and Aluminium

Recently, Professor Chris Exley from Keele University claimed that his research “provides the strongest indication yet that aluminium is a cause of ASD”. In an article originally published in the Hippocratic Post and reproduced by the Daily Mail, Exley claimed that brain tissue from five donors diagnosed with Autism Spectrum Disorder (ASD) contained “some of the highest values yet measured in human brain tissue” of aluminium. He postulated “perhaps we now have the link between vaccination and autism”.

This controversial claim comes almost two decades after Andrew Wakefield published the now discredited and retracted research into ASD and the measles, mumps and rubella (MMR) vaccine which was linked to a decline in vaccination rates and recent outbreaks of measles.

I spoke to Tom Chivers from Buzzfeed about my concerns with this paper. In his article you can read concerns from myself and eminent academics Professor Dorothy Bishop and Professor Jonathan Green, renowned experts in neurodevelopmental biology and child and adolescent psychiatry respectively.

Such a controversial claim which can affect the health of children worldwide really needs some solid supporting evidence. So, let’s take a look at the paper itself.

Aluminium in brain tissue in autism

The paper is due to be published in the Elsevier Journal of Trace Elements in Medicine and Biology in March 2018 and the accepted paper is available to read online. The authors introduce the study saying “we have measured aluminium in brain tissue in autism and identified the location of aluminium in these tissues”. The paper can be split into two sections.

Section one

The authors measured the levels of aluminium in brain tissue taken from five donors aged 15 to 50 who had all been diagnosed with ASD prior to their deaths. They took 0.3g tissue samples for four (occipital, frontal, temporal and parietal lobes) or five (they added hippocampal tissue where it was available) brain regions. They measured three replicates of each brain region using spectrometry.

The problems with the methods and data from this section are three-fold:

  1. The authors took samples from only five brains – all five donors had died with a diagnosis of ASD. There were no healthy control samples used anywhere in this study. This is a particular problem because without a healthy control, it is impossible to really say whether the aluminium levels measured in the brains of the donors is actually higher than that of a non-autistic brain. There is nothing to compare the measurements to.
  2. Importantly, the authors do not provide information on how the donors died. We have to take their word that the cause of death does not account for variation in brain levels of aluminium. An important part of the scientific publication framework is that this sort of information should be easily available to the reader. We need to be able to come to our own conclusions based on the data presented. That this information is missing is quite concerning.
  3. Finally, in some cases the three replicates from each brain region per donor did not corroborate each other. In one case there were three measurements of 0.01, 0.64, and 18.57μg/g. Similarly, another donor had measurements of 2.44, 1.66, and 22.11 and another, 1.71, 1.64, and 17.10μg/g. The authors averaged these data, however such an average does not represent any of the three measurements and is an inappropriate way to analyse these figures. In fact, such variation between measurements might even hint at an anomalous result, which requires further assessment to adequately rule out.

Section two

In the second section of this paper the authors took samples of brain tissue from the valuable Oxford Brain Bank. Again, they took tissue from a selection of donors who had been diagnosed with ASD before their death. Again, the authors failed to express how the donors died. The authors took samples from 10 donors aged 14 to 50 and stained them with a dye that specifically sticks to aluminium. The dye can then be visualised with a fluorescent microscope to see where in the brain tissue it can be seen.

Again, the issues with this section are three-fold

  1. This section also lacks the use of ‘healthy’ controls. The authors do show presence of aluminium in the tissue samples of their ASD donors but we have no indication of whether this is, in fact, more than we might see in a healthy brain.
  2. This section is also lacking a positive control – a sample that has a known amount of aluminium in it. This is important for interpreting the staining of the dye; if the dye is very sensitive then we might see loads of staining for just a small amount of aluminium. We need to compare it to a known sample.
  3. Later in their interpretation of these data the authors discuss the presence of aluminium in specific cell-types which they surmise might be lymphocytes. However, they do not use any detectable markers to propose this, they base it only on how the cells look. Cell appearance (also known as morphology) is a useful initial indicator of cell-type but it is important to confirm this using specific markers for those cells.
1-s2.0-S0946672X17308763-gr1
A figure taken from the paper which apparently shows the accumulation of aluminium in specific cell types

Overall the data from this paper is insufficient to draw the conclusions made in the Hippocratic Post and The Daily Mail and it is my concern that such reporting is irresponsible. While the two articles conclude that aluminium present in the brains of people with ASD might be caused by vaccination, there is no investigation whatever into the source of such aluminium in this study. There are no data presented in this paper sufficient to implicate vaccination in these findings. To draw the link between aluminium in the brain of people with ASD and vaccinations makes for a striking headline but the data do not support this claim and it could therefore be viewed as scare-mongering.

I am surprised that this paper was published in the Journal of Trace Elements in Medicine and Biology because the lack of control data makes the findings inconclusive. I am yet more surprised that this inconclusive data has been reported in such a paper as The Daily Mail, when such media attention can lead to parents making potentially dangerous decisions for the health of their children.

Please let me know below what you think about the reporting of this science?

How many mutations does it take to create a cancer?

Last week I wrote a post all about how we understand cancer to arise. I explained how mutations in certain genes for certain proteins can drive cancer and I explained some of the important families of proteins such as oncogenes and tumour suppressors.

But even that complicated post was an over-simplification. Sure, a single mutation in a single gene for a single protein might cause cancer, but actually we know that many cancers have several mutations. Sometimes, several hundred mutations.

Several hundred mutations

many mutations per cancer
Image adapted from this paper. A graph showing a number of non-synonymous mutations per tumour against tumour types ranging from colorectal cancer to glioblastoma. (See the text for some example figures).

The above is an image which shows just a few examples of some cancer types and the number of mutations you might expect to find in them. Recognise the word non-synonymous from last week? Non-synonymous mutations are those which cause a change in a protein. Whereas synonymous mutations are effectively “silent” (they don’t change a protein) so we tend to focus on non-synonymous ones when it comes to disease. You can read more about them here.

From the image you can see that some types of cancers can have several hundred mutations per tumour – colorectal cancer for example can have between 500 and 1200 mutations. Lung cancer can have between 100 and 200 (interestingly this is much lower for lung cancer in the “never-smoked” category which has a considerably smaller number of 25) and breast cancer between 25 and 100 mutations per tumour.

The problem

What this means is that when it comes to understanding what drives cancer we need to know how many of these mutations are actually relevant to the disease. Do they all contribute to “driving” cancer or are some of them “passenger” mutations? If only a few contribute, then how many?

Often, we treat cancer by designing drugs which target specific proteins. If we suspect a cancer is “driven” by an overactive protein then we can treat patients with an inhibitor of that protein – something that blocks the protein’s function. But for us to do this, we need to know which mutation, potentially hundreds, is actually relevant. It’s like trying to find a needle in a haystack.

The study

The paper published in October 2017 in Cell is an analysis of 7,664 tumours across 29 different tumour types. They looked at all of the mutations each of those tumours had and focussed on the non-synonymous ones, the ones that actually change the proteins inside the cell. But they knew that even with protein changes, not all of the mutations present actually cause cancer. They wanted to establish how many might be responsible for the disease.

“Normal” cells acquire mutations all of the time but this happens at random so sometimes it will affect proteins and sometimes it won’t –  we get a reliable mixture of non-synonymous and synonymous mutations that are all mostly harmless even if there is a change in a protein. This is useful information because we can establish an “expected” ratio between the two. In cancer cells, the mutations that change proteins and allow a cell to survive and grow more than it should (i.e. the mutations that drive cancer) are passed on to more cells as the cancer cells replicate. This means you get a skewed ratio in cancer cells – more non-synonymous mutations than synonymous ones. By figuring out how many more mutations there are than we expect from the “normal” cells we know that those are the ones allowing cells to replicate in an uncontrolled way – therefore those are the ones that are driving the cancer.

And that’s exactly what the authors of this study did.

What they found

What they found was that although some cancer cells have hundreds of mutations, only a few of those actually drive the cancer. The average number of cancer drivers per cancer type is just four and the numbers only range from one to ten. So, we can narrow our focus to between one and ten mutations per tumour. We just need to figure out which ones.

This is phenomenally useful information because we can then build further on this knowledge to understand which mutations are the common driver mutations and focus our drug development on those specific mutations. We have a significantly smaller haystack and we can start to really get very specific with our cancer therapies! The better our understanding of how cancer really works, the more complete our understanding of all of the complicated processes involved, the better able we are to design clever therapies that really benefit patients.

What drives cancer?

When it comes to understanding cancer, getting our heads around the complicated process of how it forms in the first place is vital. When we know what drives cancer then we can figure out how to diagnose it and ultimately, how to treat it.

So far, research has shown us that the vast majority of cancers are caused by some level of genetic mutation. This is something we’ve known for a long time and something we use in order to treat different types of cancer. For example, a reasonable proportion of breast cancer patients have a mutation in the gene for a particular protein called HER2 making this protein more abundant than it should be and allowing cancer cells to grow and grow. So scientists have designed a therapy that blocks, or inhibits, HER2 – a drug called Herceptin. The cancer cells have been relying on HER2 for their growth, they’ve becomes entirely dependent on it which means when we treat patients with Herceptin, HER2 activity is reduced and the cancer cells die.

Another thing we know about cancer, is that it’s hugely genetically unstable. This means cancer cells acquire loads and loads (sometimes many hundreds) of new mutations as the disease progresses. What is important is to understand which of these many mutations are actually fundamental to the survival of the cancer. Which ones are truly driving the disease, allowing it to survive and progress and spread?

This can be particularly complicated for us to understand because a mutation in one gene can do lots of different things.

Genes

First things first, let’s define what a gene is. When it comes to DNA we can think of an organism’s entire genome like a recipe book. The entire human genome book allows our cells to work together to cook an entire human. If we were cooking a really complicated meal like Christmas dinner we would need to cook all the different components and bring them all together to make the meal. You have to cook the main dish and all the side dishes, your carrots and parsnips and brussel sprouts, maybe two kinds of potatoes and a soup for the starter. Every person’s Christmas dinner might look slightly different but we all agree it’s a complicated meal and there are lots of components. The human body is also pretty complicated and is made up of components. A human body is made up of organs, which at the most basic level are made up of cells. Your cells are kind of like the ingredients for each dish in your Christmas dinner. Your organs are kind of like those individual dishes. They all come together to make the whole human. So, in order to make such a complicated thing, we need lots and lots of recipes. Within the human genome we have 23 pairs of chromosomes, which we might think of as chapters of the recipe book. Within each chapter are individual recipes, or genes, which allow cells to make for individual building blocks (ok, ok, I’m mixing my metaphors…).

The basic building blocks in the human body are cells. Cells come together to make our organs, and organs come together to make a human. But our cells need to do specific jobs depending on what kind of cell they are; they need to communicate with one another within the organs they are a part of. This is what makes our heart cells contract all together to pump blood around the body or it allows all of our lung cells to know that they’re supposed to be lung cells. This communication is done through proteins, which can pass signals along complicated pathways transmitting the message from protein to protein and giving each cell instructions on how to function. A single gene is a recipe for an individual protein. Each gene is made up of many thousands (depending on the protein size) of what we call bases which are signified by the letters A, T, C and G. There is an extra layer of complexity as the code that makes up a single gene is organised into groups of three letters each group referred to as a codon. Each codon codes for a single amino acid and these amino acids build up in long sequences to eventually form proteins.

DNA to protein
In order to convert the genetic code the cell must convert DNA into an intermediary molecule called RNA. This can then be translated into protein. Each ‘codon’ (three letters) is a code for one amino acid which build up in sequences to make proteins.

A mutation is change in this code. And that tiny change can have a number of outcomes.

Ultimately – the ones we’re worried about are those that change how particular proteins behave.

How do protein mutations drive cancer?

On a very basic level, proteins basically control everything. They regulate which cells go where, how big cells grow, how much cells replicate and how cells function within their organs. Some proteins are crucial to very specific pathways related to cell survival and cell replication (referred to as proliferation). These proteins we refer to as oncogenes or tumour suppressors. They carefully regulate how cells grow and survive. If these pathways become deregulated, cells can grow out of control and survive when they shouldn’t. This is how cancer forms. These pathways, and therefore these particular proteins, are fundamental in driving cancer. Many of the most commonly mutated proteins in cancer are either oncogenes or tumour suppressors. But what exactly are these proteins?

An oncogene is a protein which promotes cell growth and cell survival. In normal cells these proteins are carefully regulated. They become activated and they pass on the message to tell the cell to grow or survive and then they get switched off until they are next required. Oncogenes can be mutated in such a way that their activity is increased – either there are loads of copies of them all passing on the “grow and survive” message at once, or they are unable to be switched off and they are constantly telling the cell to grow and survive. In this way, a mutation in an oncogene can drive cancer.

A tumour suppressor is one of those proteins that helps regulate the growth and survival pathways. Tumour suppressors can switch oncogenes off, or they can oppose them by sending the opposite message. They serve to slow down the “grow and survive” message and keep our cell growth and survival finely tuned. In this way, when functioning properly, tumour suppressors prevent cancer. A mutation that blocks these proteins from doing their job will therefore promote cancer.

Oncogenes
This is adapted from my PhD thesis. Here are two pathways with the various proteins that ‘transmit’ the signal through a cascade of activation. One pathway leads to cell replication (proliferation) and the other to cell survival. Some of these proteins are particularly crucial in promoting these pathways – PI3K and Ras, for example, are oncogenes. Some of these proteins are crucial in regulating or slowing down these pathways – PTEN, for example, is a tumour suppressor.

Different types of mutation

This complexity of the genetic code means there are number of different ways in which a gene can be mutated.

We can split these into two broad groups – synonymous and non-synonymous. Synonymous mutations are ones where the mutation doesn’t lead to change in the sequence of amino acids in a particular protein. They are effectively silent mutations because they don’t change proteins.

But many gene mutations are non-synonymous; one way or another, they cause a change in the protein produced by that gene.

Of these we can have:

Missense mutation is a mutation where one amino acid is switched out for another. The importance of that particular amino acid to that particular protein designates whether or not this change has a big impact on the protein function. It can increase activity of an oncogene, or it can prevent an active oncogene from being switched off. Alternatively, it can block the action of a tumour suppressor.

Nonsense mutation is a mutation which leads to termination of the protein production at that position in the code, leading to a shortened or truncated version of the protein which may severely alter the ability of that protein to function normally. A truncated tumour suppressor, for example, isn’t able to switch off cell growth and might promote cancer.

Insertions/deletions occur when a few letters are added to or removed from the code which can completely alter the entire remaining sequence leading again to protein truncation. Large deletions can remove entire genes preventing the production of that entire protein. This can include duplication mutations when a particular region of DNA is duplicated which may enhance or reduce the activity of a protein.

Large-scale mutations in chromosomal structure – these can be very complicated including the amplification of entire genes giving you far too many copies of a particular protein. They can also create entirely new proteins by fusing two separate genes together.

What does all this mean?

Hopefully it’s clear that the variety of mutations and proteins involved in driving cancers is quite complicated and that makes understanding cancer difficult. However, we might also see opportunities for using knowledge to treat cancers. If we can establish how frequently a particular cancer is cause by an overactive oncogene then we can start to find treatments that block or inhibit those oncogenes. These are strategies that have led us to discover drugs like Herceptin, which blocks the oncogenic HER2 receptor I talked about at the beginning of this post. But it also means we need to fully understand which mutations drive cancer in order to establish treatments for them.

Next week I’m going to talk about a particular study that looked at these driver mutations that was published just this month. The researchers wanted to get a more complete picture of how many proteins we might need to target in order to effectively treat specific types of cancer and what they found was really interesting.

For now, I’d love some feedback on this post because it’s quite science heavy – if there’s anything I could have explained better or in more detail then let me know in the comments and maybe I can write more on these topics in the future.

 

*edited to correct some of the HER2 science – thanks to my good friend Dr Jenny Smith-Wymant for pointing out my mistake.

Will we ever cure HIV infection?

So far on this blog, I’ve talked a lot about cancer. I’ve been working in cancer cell biology for a number of years, and it’s an area that a lot of people are interested in, so it makes for a sensible topic. By training, though, I am a cellular and molecular physiologist. I’m interested in how all cells work; they don’t have to be cancer cells.

The human body, and cells in particular, amaze me. I love learning the intricate details of exactly how our cells work on a molecular level, but I love how we can identify little changes that might be useful in understanding disease and treatment even more.

Earlier this year I came across an interesting study on treating individuals infected with the Human Immunodeficiency Virus (HIV). HIV is a complicated virus and has long been a source of fear. But by understanding the science of the virus we can look to treat infection and perhaps dispel some of those fears. So here’s a bit about HIV, and then a bit about a cool study.

HIV

HIV is a type of virus that we call a retrovirus. A retrovirus carries its genetic material as a single strand of RNA, instead of the double-strand of DNA we use as humans. To transmit its genetic material the virus needs to construct DNA, which it does inside a host cell. In the case of HIV, the host cells are cells within the human immune system – white blood cells called helper T cells.  Once an HIV molecule (a virion) gets inside a human host cell, it uses an enzyme within the virus to transcribe a piece of its RNA into DNA. It then slots that DNA into the host cell genome. It can remain dormant for many years but eventually the host cell machinery produces many, many copies of this piece of DNA, releasing more HIV virions into the blood stream. Essentially, the virus hijacks the host cell’s normal processes in order to make copies of itself.

Picture1
The life cycle of HIV: the virus enters a host cell and releases its RNA which is transcribed into viral DNA. Viral DNA is integrated into the host cell DNA where it can lie dormant for many years. Upon activation, the host cell produces viral RNA allowing the generation of new virions which are released into the blood and are able to infect new host cells.  Image taken from Encyclopaedia Britannica.

The HIV virus does not need all the machinery required for its own replication therefore the HIV genome is tiny, consisting of only nine genes. By comparison, the human genome consists of around 20,000 genes!

Treating HIV

These days we are pretty good at treating and managing HIV infection. HIV infection isn’t currently curable, but we’re so good at treating it that many patients live a long, healthy life while carrying HIV. There are many caveats to that, including political problems related to getting treatment to all the parts of the world that need it.

The very first treatment we produced for HIV was an inhibitor that blocked the action of a protein important in converting the virus’ RNA into DNA, so preventing its subsequent replication and spread throughout the body. Since then we’ve designed different types of antiretroviral therapy (ART) which target different processes within the virus’ lifecycle. We can stop the virus getting into a cell, stop its RNA converting to DNA, stop that DNA integrating into the human genome and stop the virus from assembling and breaking out of the host cell. The problem is, once the virus is integrated into the host cell DNA it can lay dormant, untouched by inhibitors even when the viral load is reduced in the patients’ blood. We have to maintain treatment to prevent this dormant DNA from activating and the virus spreading around the human body. It is possible, too, for the virus become resistant to each therapy meaning careful assessment of the patient and modification of the therapy accordingly. In this way, we can manage the infection long-term. But what if we could figure out a way to target the dormant virus sitting in host cell DNA?

New study

Picture2
Title and authors of the new study published in September 2017 in PLOS Pathogens

A new study published in September aimed to do exactly this. Earlier research had shown that a particular type of drug can ‘activate’ the host-cells which hold dormant HIV DNA. These ‘activated’ cells produce lots and lots of RNA for the virus which “kicks” the hiding virus back into action. Using this principle, the standard anti-retroviral drugs are then able to “kill” the virus specifically targeting this pool of dormant virus that standard therapy alone is unable to target. This strategy is referred to as “kick and kill”. While other researchers have studied this principle before, the effect on host-cell activation has been minimal which means only a small proportion of cells holding dormant HIV can be targeted. The larger this proportion, the closer we can get to activating and killing every cell holding dormant-HIV and the closer we can get to curing HIV infection.

In this study the authors used a synthetic compound, SUW133, which would they hoped might activate more host cells and therefore be more successful.  Their initial work looked at the effectiveness of the compound in cells taken from six patients. They showed that control treated cells (treated with only media) had relatively few RNA molecules from the HIV virus whereas when they took those same patient-derived cells and treated them with the SUW133 the number of viral RNA molecules increased significantly. This indicates that SUW133 does, indeed, activate the dormant virus and kicks the host cells into action.

Picture3
Data taken from the paper. Comparison of control treated human patient cells with SUW133 treated human patient cells. Samples derived from the same individual patients are connected with a line showing the increase in activation of cells per patient when treated with SUW133.

They then wanted to look at what would happen in a living organism, so the researchers had to infect humanised mice (mice genetically modified to express human genes) with HIV. They did this for 3-4 weeks, after which they treated the mice with antiretroviral therapy for 2-5 weeks. Once they were sure that only dormant HIV would remain in the mice, they treated them with their special synthetic compound and looked in the blood for evidence of HIV-host-cell activation. In mice treated with a control (inert) compound they found relatively low proportions of specific cell types (CD3+/CD4+ and CD3+/CD4-) that expressed a marker for activation (CD69) however upon treatment with the active compound (SUW133) the proportion of “activated” CD69 positive cells rose dramatically.

Picture4
Data from the paper. The proportion of  activated (CD69+) blood cells in the blood of HIV positive mice is much higher in mice treated with SUW133 compared to control treated mice.

What’s more, the researchers showed that a small proportion (around 25%) of these activated cells were dead or dying which shows promise for the additional treatment with standard antiretroviral therapy to fully “kill” the virus.

Overall the authors have shown that they can activate dormant HIV which they hope will give us the opportunity to kill this currently untreatable pool of the virus and one day give us a cure to HIV infection.

What’s clear is that this is still early days – the researchers need to fully establish the effect of this “kick” strategy when combined with the “kill” treatment (ART). However, it is incredibly promising that we might someday be able to activate host-cells carrying dormant HIV genetic material so that we can remove those cells entirely. If we are successful, we could go from long-term HIV infection management to HIV-infection cure.

 

Science or Pseudoscience: Ancient Chinese Calligraphy Ink and Cancer Treatment

When it comes to reading about cancer cures in the media it is usually good advice to be pretty skeptical. Sometimes the story lends well to skepticism – like this one recently covered in a few outlets where it was proclaimed that scientists had found a cure for cancer in ancient Chinese calligraphy ink. It’s a story that has everything – including lasers. Here is a romantic idea that the cure for cancer might lie in an ink that is produced from plants and has been handed down between generations as a way to share writing and art. The proposed treatment is billed as non-invasive and specific only for cancer cells – could this really be science?

I was particularly suspicious when I saw that the representation of the story on Natural News, a prominent proponent of pseudoscience, looked a lot like the story on Science Alert which is typically more of a reliable source on science stories. So I did some digging into the data for this finding.

The paper that was covered in the media is titled “New Application of Old Material: Chinese Traditional Ink for Photothermal Therapy of Metastatic Lymph Nodes” and was published in the journal ACS Omega in August 2017. The researchers were based in Shanghai distributed across various institutes at different hospitals or universities. They had been working on a relatively new cancer therapy called Photo-thermal Therapy (PTT).

Photo-thermal therapy (PTT)

In cancer treatment, PTT is the use of specific types of nanoparticles to generate cell damaging heat specifically at the tumour sites. Scientists take a material that can be stimulated with light – typically infra-red – causing the generation of heat and subsequent tumour cell death. The problem is that many of these nanoparticles are toxic or expensive so in order to make the treatment as efficient and effective as possible, we need to find just the right material.

Hu-Kaiwen

Hu-Kaiwen (Hu-ink) is an ink that has been used by calligraphers in China for hundreds of years. It’s derived from plants, is mostly made up of carbon and it is black in colour which the materials used for PTT tend to be. So scientists got to wondering if it might be useful for PTT.

Firstly the authors of this study looked at the stability of the ink. They diluted it in different things including water and saline and made sure it was stable when stored over time. They looked at the structure of the ink and noticed that it typically forms small aggregates of 20-50nm in diameter – nanoparticles. They confirmed that the core component of the ink was carbon and they stimulated different concentrations of the diluted ink with infra-red lasers and tested the temperature. They found that Hu-ink was more efficient at converting light into heat than most other PTT materials reaching temperatures of 55°C after five minutes irradiation.

Hu-ink for PTT in cancer cells grown in the lab

In research we use cell models of cancer which we refer to as cancer cell lines. These are cells that have originally been taken from patients with different types of cancer but are grown and stored artificially in the lab. We use them so we can test things out on cells that behave like cancer but aren’t in a human body before we move on to doing tests in living organisms. The researchers in this study used some cancer cells originally derived from colon (SW-620) and colorectal (HCT-116) cancer. First they treated the cells with just the Hu-ink and the cells tolerated it really well proving that the ink solution itself wasn’t toxic. Then they treated the cells with the ink and combined it with irradiation with the infra-red laser.

Hu-ink
HCT-116 cells treated with Hu-ink and infra-red compared to HCT-116 cells treated with infra-red only. Image modified from the paper.

The image above is part of a figure taken from the paper itself. There are three images of HCT-116 colorectal cancer cells. In the top image, the cells were treated with only the laser for five minutes. They were not given any Hu-ink. In the middle image the cells were treated both with the Hu-ink at a medium dose for two hours and the laser for five minutes. In the bottom image the cells received a high dose of the Hu-ink for two hours and the laser for five minutes. The red cells are cells stained with a marker of cell death. The green cells are living cells. You can also see that the dead cells are much smaller than the living cells. In the top image where cells did not receive any ink – all of the cells are alive. In the bottom image where the cells had a high dose of ink – all of the cells are dead. In the middle image with a lower dose of the ink, there are some living cells and some dead cells. In other words – when you treat cancer cells with both Hu-ink and infra-red, the cancer cells die.

Hu-ink for PTT in mice

The researchers wanted to be sure that this technique was safe in living organisms and that it was able to kill cancer cells in living organisms. So they took mice with cancer of the lymph nodes and injected Hu-ink into the tumour which they then irradiated with infra-red. After an allocated treatment time, they removed the tumours from the mice and measured them.

Hu-ink2
Lymph node tumours taken from mice treated with Hu-ink and infra-red versus control treatments. Image modified from the paper.

For this experiment they had three control conditions – NS (the tumours were injected with saline), NS plus laser (the tumours were injected with saline and treated with a laser) and Hu-ink (the cells were injected with Hu-ink). They had one test condition – the Hu-ink plus laser. The test condition is the only one that the authors predicted would have an effect on tumour size. And that’s exactly what they saw. Lymph node tumours treated with Hu-ink and infra-red were significantly smaller than the control conditions. They also saw that the surrounding tissue wasn’t damaged suggesting that the treatment is safe.

Study conclusion

This study is a proof-of-principle study. The authors have shown that this ink can be used in PTT therapy with a positive effect and in a safe way in mice and in the lab. It is a very small scale study and it is a single study. It needs replicating before we can be confident in the result and it needs to be studied on a larger scale and have many more safety tests before we could begin to think about using it in patients. But it is a really promising study. It takes a treatment we already use and aims to make that treatment safer and cheaper and more available to patients. It is a non-invasive treatment and it can be used in ways that reduces tissue damage to healthy tissue while targeting cancer cells for cell death. It’s a great example of some really cool science, using materials that have been available for many years and applying them to modern techniques.

It might seem far-fetched to say ancient ink can help us treat cancer, but really it’s just cool science!

Cool science: using Zika virus to treat cancer?

Throughout the last two years there has been a great deal of news on the Zika virus – a virus spread by mosquitoes which was first identified in 1947 in Uganda. In a normal healthy adult Zika fever causes relatively mild symptoms or even none at all however the 2015-2016 Zika epidemic gave rise to widespread concern due to its propensity to cause microcephaly and brain defects in babies infected with the virus during development. The epidemic was declared over in late 2016 although there are still travel warnings to certain areas where the mosquitoes known to carry the virus are prevalent.

But research into this particular virus highlighted an interesting trait that we might be able to take advantage of – Zika has a preference for stem cells.

Zika and stem cells

The reason Zika virus is particularly dangerous in developing babies is that the virus causes damage in stem cells in the brain. A stem cell is a cell that isn’t yet programmed. They’re really important in development because when you create a baby you start with one sperm cell, one egg cell and these cells needs to combine, proliferate and then differentiate into all the different types of cell within the human body. Stem cells are unique cells that can be programmed or ‘differentiated’ into all sorts of different types of cells. Once a stem cell has differentiated it can’t turn back into a stem cell – a differentiated cell is committed to only ever being that cell type. The adult body has very, very few undifferentiated cells but a developing foetus has plenty. This explains the risk of Zika infection during pregnancy as Zika has been shown to target neural progenitor cells – a type of undifferentiated cell in the brain – that might lead to the microcephaly seen in babies infected with the virus during their development.

480px-Zika-chain-colored
Crystal structure of the Zika virus

Zika and brain cancer

Cancer stem cells are quite a complicated thing that I’m not going to try and do justice in this post because it’s a topic that deserves its own post. Scientists believe that some cancers do have associated cancer stem cells. How exactly cancer stem cells might contribute to cancer progression is far from fully understood. However, we do believe that the presence of cancer stem cells might contribute to cancer therapy relapse. This is particularly concerning in glioblastoma – an aggressive form of brain cancer, which has poor survival rates despite our best efforts. Without treatment, median survival is around 3 months from diagnosis. With treatment we are able to extend that survival to 12-15 months however the cancer usually recurs. Scientists believe this recurrence is all down to the presence of cancer stem cells.

The study

So here’s the clever part. Cancer researchers know we have a problem in treating glioblastoma. They also realised that Zika virus is a relatively mild virus, which attacks stem cells. The adult brain doesn’t really have stem cells – unless the adult has glioblastoma. Cancer stem cells in the brain lead to cancer relapse; Zika attacks brain stem cells. Maybe we can make use of these two pieces of information.

So, scientists did some experiments. Firstly, they took some glioblastoma cells from patient tumours and they grew them in a dish in the lab. Then they infected them with Zika virus. They looked at either glioblastoma differentiated cells or glioblastoma stem cells. And they looked at the infection rate. Over 48 hours, over 60% of the stem cells were infected and this increased over time as the virus spread. The differentiated cells were infected too but not as much. What was especially interesting was that the stem cells infected with the virus had severely reduced ability to multiply and they had an increase in cell death. This was specific to only the stem cells and didn’t affect the differentiated cells. The virus kills cancer stem cells and prevents them from spreading.

Next the scientists took some patient tissue samples – this allowed them to look at a whole mixture of cells in a slightly more normal context without having to infect patients. They infected the tissue samples with Zika and saw that cancer samples were infected successfully and the virus only hit the stem cells and not the other cell types in the sample. They also looked at some brain samples from epilepsy patients and the virus didn’t infect them showing that the virus really is specific for stem cells!

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Glioblastoma tissue sample (from the paper)

 

Finally, they used the virus to treat mouse models of glioblastoma. They took mice with glioblastoma tumours in the brain and infected them with the virus. They saw that the tumours were much smaller and the mouse had improved survival when they were infected with the virus compared to control treated mice. They went on to show that they got an even better effect when combined with other glioblastoma treatments.

Benefits

Current treatments have two problems when it comes to glioblastoma. Firstly, the cancer stem cells make recurrence almost inevitable. This could drastically improve average survival times. Secondly, all brain cancer treatments have to be able to cross the blood-brain barrier in order to get through to the cancer cells. The blood brain barrier is an important way to keep things out of the brain where they might cause damage but it also serves as a way to keep brain tumours trapped in and harder to treat. Zika is great at crossing the blood-brain barrier.

What next?

We’re still such a long way from this being a useful patient treatment. In order to use this as a treatment we need to modify the virus in such a way that it will not spread from person to person and it will not cause the patient any harm. Currently virus work is always done in very specialised laboratories with expert training on how to prevent spread and with many, many precautions. If it were to be used as a therapy we’d need lots and lots of precautions to make sure it were safe. So far this has only been done in lab grown cells (albeit ones taken from patients mouse models of cancer. But it’s incredibly interesting research and a great example of how cancer research is so quick to develop and understand how we can take advantage of what we know about the disease and use that to treat it.

 

Please let me know in the comments if you’d like to see a post on any of the topics from this post – Zika virus, glioblastoma, stem cells?

If you found this interesting, please share it with three other people who might find it interesting too! Sharing cool cancer research gives us all a little more hope!

 

Image credit for the crystal structure of Zika: By Manuel Almagro Rivas – Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=47941048

Cool Science: taking advantage of cancer cell biology for cancer treatment

One of the big problems when it comes to treating cancer using drugs is that these drugs flood the patient’s body and cause detrimental side effects when they reach areas other than the malignant tumour.  Cancer cells are derived from our own healthy cells – they’re hard to target specifically without also hitting our healthy cells. A lot of research goes into trying to get around this problem. I came across a particularly interesting study that I thought I’d share here.

Earlier this year Sofie Snipstad et al. published a paper in Ultrasound in Medicine & Biology titled “Ultrasound improves the delivery and therapeutic effect of nanoparticle-stabilized microbubbles in breast cancer xenografts”. This paper was particularly cool because of the rationale behind it. Nanoparticle delivery of drugs is something scientists have been working on for a little while. The premise is that you take your drug and you wrap it up inside a small protective bubble allowing the drug to travel to a specific site before it is released. Due to a quirk of cancer biology, this is particularly great as a cancer therapy. When tumours grow, they start to form their very own blood supply – only the blood vessels that they grow are more leaky than normal blood vessels. They allow slightly larger molecules to pass through from the blood vessel and into the surrounding tumour.  This means we can use nanoparticles to deliver cancer drugs specifically into tumour sites by allowing the particles to travel through these leaky blood vessels. But then we hit another problem – if the tumour blood supply doesn’t reach the very depths of the tumour then the particles are too big to get all the way through. You can treat the edges but not the very centre of the tumour. So, this paper worked on a special combination – they took a bunch of nanoparticles containing chemotherapy and they bundled them up together into microbubbles that can travel around the blood system easily and safely until they reach the tumour site. Then, the researchers used a focused shot of ultrasound to break up the bubbles and release the nanoparticles. This also served to allow gentle tissue massage by the ultrasound to allow the nanoparticles to distribute further throughout the tumour. Once in the tumour, the cancer cells start to take up the nanoparticles and inside the cell the drug is released and can kill the cancer cell from the inside.

Picture1Image: figure from the paper

Any microbubbles that weren’t in the tumour site and therefore not exposed to the focused ultrasound could be easily cleared from the body without releasing the drug which means the only cells targeted by the therapy are the cancer cells. This means we can hit the cancer cells with a higher, more toxic dose because the healthy cells are not going to be hit with the same dose.

The researchers in this paper were testing the optimal way of doing this in mice suffering with triple negative breast cancer – one of the more aggressive forms of the disease – with positive results. The mouse tumours took up the drug 2.3x better and there was no tissue damage identified. All of the tumours either regressed or else the mice went into complete remission. The authors described this as a “promising proof-of-concept study”.