Fatbergs Block Cancer Defences

 

Most people are aware that being seriously overweight is a health hazard — and it’s a big one because nearly 2 billion adults are overweight or obese. Obese people are 80 times more likely to get type 2 diabetes than those of normal weight, they’re more likely to suffer from heart and blood vessel disease and they have an increased risk of cancer. In fact about half of some types of cancer are caused by obesity and in the UK more than 1 in 20 cancers are due to excess weight — making it the second largest preventable cause of cancer (smoking, of course, being the first).  Indeed, new figures from Cancer Research UK published a few days after I wrote this piece tell us that now obese people outnumber smokers two to one and being overweight causes more cases of certain cancers than smoking.

As you know

Obesity is associated with abnormal levels of hormones involved in growth (e.g. insulin, oestrogens and leptin). It’s generally thought that their raised levels also favour cell proliferation and tumour growth. Nevertheless, despite the figures showing a clear link, it’s been a slow business to unearth the molecular links between obesity and cancer. And that knowledge is, of course, essential if we’re to come up with ways of interfering with the process.

In Obesity and Cancer we noted that two things happen as obesity develops: the number of fat (adipose) cells goes up but they also grow bigger (i.e. the fat cells themselves are fatter).

This causes a knock-on effect that is even more serious: the fat cells attract other cells from the circulation and this cellular cooperative releases signalling proteins that can drive tumours.

In obesity abnormal signals from fatty tissue can combine with others arising from perturbed metabolism to help cancers develop.

With that background we described in Isn’t Science Wonderful? Obesity Talks to Cancer the discovery that cells recruited into the tumour neighbourhood can talk directly to the tumour cells. They do this by releasing the messenger leptin — a hormone made by adipose cells that stops us feeling hungry.

The cellular ‘groupies’ that make leptin are fibroblasts – part of the supportive framework of cells and tissues, so they’re ‘cancer-associated fibroblasts’— rather than fat cells, but that’s slightly by the by.

Now comes another piece of the jigsaw, courtesy of Xavier Michelet, Lydia Dyck and colleagues from institutes in Boston, Kentucky and Ireland, who have shown that one upshot of obesity can damage our anti-cancer defences. It does this by taking aim at natural killer cells (NK cells) — a sub-group of white blood cells (lymphocytes) that are a key bit of our immune system when it comes to destroying tumour cells. NK cells attack tumour cells directly, making holes in their outer membranes and essentially blowing them up.

Obesity paralyses immune cells. The two images are of immune cells from (left) lean and (right) obese individuals. The cells were stained with a fluorescent indicator that detects fat molecules. White bar = 10 microns (i.e. one 10 thousandth of a metre). From Michelet et al. 2018.

Michelet and colleagues showed that circulating free fatty acids (FFA) are taken up by NK cells. As the levels of FFAs are raised in obese individuals, their NK cells accumulate FFAs. The photo above shows how abnormal these fat-loaded cells look and it’s no surprise that their metabolism gets upset. Critically for their anti-tumour activity, this disruption cuts production of the proteins that target tumour cells (perforin and granzymes).

So at last we have a clear molecular link between obesity and cancer: the raised levels of FFAs push a metabolic switch in NK cells that blocks their ability to kill tumours cells — so a major repressor of tumour growth is overcome.

Reference

Michelet, X. et al. (2018). Metabolic reprogramming of natural killer cells in obesity limits antitumor responses. Nature Immunology 19,1330–1340.

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3D Tumour Printing

 

Having for decades thought of ‘printing’ as an operation that produces an image in two dimensions (I know: physicists please don’t write in, you know what I mean) I’ve had a lot of difficulty grasping the idea of three-dimensional printing. It’s presumably an example of ‘old fogey set in ways’ because in fact the idea’s not that difficult.

How’s it done?

A computer-controlled printer deposits successive layers of material to build the 3D shape specified by the computer. A number of methods have been developed but so precise has the technology become that, using computer-generated models, 3D geometries of almost unlimited complexity can be produced.

The methods, referred to as additive manufacturing, have developed to the extent that they are now used in heavy engineering and architectural design, for model railways and prosthetic implants. In the medical field you can now obtain patient-specific implants, based on anatomical data from the patient acquired by making 3D pictures of the target (e.g., by CT scans), for pretty well any item from jaws to ankles. The product is a bespoke implant, tailored for the individual patient, and they must be a fantastic boon for surgeons.

Now for the really amazing bit …

That’s a stunning example of different areas of science converging to produce completely novel strategies but what got me thinking about 3D printing was a recent paper that had the extraordinary juxtaposition of bioprinting, human tumours and chips in its title! The stars who’d done this remarkable work were Dong-Woo Cho, Sun Ha Paek and colleagues in South Korea. The problem they’d tackled was to come up with a model system for the most common brain cancer, glioblastoma, for which the five-year survival rate is less than 5%. Model systems in which cells from a patient’s tumour are implanted into mice have been developed for brain tumours but so far these have been poor predictors of treatment response.

It’s been recognized for some time that the physiological locale of tumours (the microenvironment) is critical to their behaviour (see Trouble With the Neighbours, Mosaic Masterpieces) and the Korean groups tried to reproduce that more closely by using 3D printing. They added to the cancer cells an extract of animal brain tissue together with endothelial cells (that line blood vessels) and gas-permeable silicone. Deposited on glass, that creates a more realistic glioblastoma microenvironment that includes being able to mimic the variations in oxygen levels that occur in tumours.

The result was that not only did the tumours grow but within a couple of weeks it was possible to test their responses to various drug combinations.

3D tumour printing on a chip to test drug responses in vitro. The first step is to remove cells from a patient’s brain tumour (a glioblastoma — GBM) and to mix them with a ‘bioink’. Several other ‘inks’ are added to mimic the natural environment of the tumour. The mixture is printed onto a glass slide and grown for two weeks before testing combinations of candidate drugs to inform the treatment plan for the patient. From Yi et al., 2019.

A major step

This tumour-on-a-chip method promises to be a significant advance in customizing treatments for glioblastoma. What’s more, its use will not be limited to brain tumours. However, as always with scientific progress, it’s not the final deal. For one thing this system cannot reproduce an immune response and we know that is a critical modulator of tumour progression.

Even so, it represents a quite astonishing marriage of scientific approaches to the problem of cancer treatment.

Reference

Yi, H.-G. et al. (2019). A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy. Nature Biomedical Engineering, 18 March.

Gomez-Roman, N. and Chalmers, A.J.   Patient-specific 3D-printed glioblastomas. Nature Biomedical Engineering (2019).

Joining Europe

 

Readers will not need me to remind them that Britain has always been part of Europe — at least since the days of the supercontinent Pangea when all the landmass of the earth was glued together in one lump. That was about 335 million years ago and it began to break up some 175 million years ago.

Modern continents in the Pangea jigsaw puzzle.

Britain (and let’s not forget Ireland) was too small to be affected by such massive tectonic events and so remained joined to the continent until after the last ‘Ice Age’ when, only a few thousand years ago, a bit of a flood covered the separating plain and, woohoo, we had the English Channel. And how handy that has been — not least because if you’re into paleontology you can dig up bits of Siberian woolly mammoths, who walked here before the flood, without trekking to Asia.

Après le déluge

Thus almost, allegedly, Madame de Pompadour when, in 1757, matters French took a turn for the worse in the Seven Years’ War (getting thumped by the Prussians in the Battle of Rossbach). Things were graver than she thought because, long before that military setback, the French language had been going downhill — slightly surprising because, after the Norman Conquest of Britain you might have thought French was set to become the world’s lingua franca, so to speak. But it turned out that over the next 300 years the English spoken by the occupied peasants — albeit absorbing lots of French along the way — gradually evolved into the language of England on its way to becoming a world-beater. That’s what the authors of “1066 and All That” would describe as a ‘Good Thing’ — especially for us dumb Brits because we don’t have to remember which of three genders might apply to our cat.

But, sad to relate, we’ve drifted away from the continent in less happy ways, one of them coming to light only over the last few score years as our life span has increased dramatically and cancer has come to the fore as a major cause of death.

What’s the problem?

For many years a little publicised and rather disgraceful fact has been that UK cancer survival rates lag somewhere around 10% behind those for pretty well every other European country. In other words, for the last 50 years you were significantly more likely to die of cancer in the UK than in most European countries. In 1970 that ‘significantly’ was about 50% but since the late 1980s the difference has gradually come down to between 5% and 15% depending on the cancer and the country of comparison.

Breast cancer mortality rates for Britain and the European Union (age standardized). This shows the trend since 1970 and the predicted rates for 2019. The trends are similar for all cancer types taken together. From Malvezzi et al., 2019.

What’s the cause?

The cause of this lamentable state of affairs is a combination of factors: diagnosis at a later stage, perhaps because Britons are disinclined to seek medical advice, and quite wide variations in the standard of treatment across the UK being two. The latter has given rise to the sad reality of the post-code lottery. In real terms this means you’re twice as likely to die of cancer in Liverpool as in Kensington. It’s a formal possibility that in the UK we suffer from types of cancer that are more aggressive — i.e. more difficult to treat — than occur in Europe but there’s no evidence for this. In others words the problem is of our own making: how we look after ourselves and the efficiency of our health system.

Just in passing we should note this isn’t only a British problem. Not dissimilar variations occur, for example, across the states of America: Kentucky bad, Utah good (186 deaths per 100,000 population versus 120). But back to Europe.

Things are getting better!

All is not lost, however, as the most recent study has shown. For the EU the rates have been falling since the early 1990s and the picture is similar for the UK but with a more dramatic drop (hooray!) albeit starting from a much higher rate (boo). What’s more, the pattern is similar for all cancers taken together. Thus not only has the gap continued to narrow since 1970 but the prediction is that by 2019 the UK will well and truly have joined the European Union in that our overall cancer survival rate will be the same as for most EU countries. This good news is exemplified by the data for breast cancer shown in the graph.

Hello … and goodbye?

So at long last on the cancer front we are about to join Europe — quite possibly at precisely the moment we brexit therefrom. How’s that for irony?!

Reference

Malvezzi, M. et al. (2019). European cancer mortality predictions for the year 2019 with focus on breast cancer. Annals of Oncology, mdz051

Secret Army: More Manoeuvres Revealed

 

I don’t know about you but I find it difficult to grasp the idea that there are more bugs in my body than there are ‘me’ cells. That is, microorganisms (mostly bacteria) outnumber the aggregate of liver, skin and what-have-you cells. They’re attracted, of course, to the warm, damp surfaces of the cavities in our bodies that are covered by a sticky, mucous membrane, e.g., the mouth, nose and especially the intestines (the gastrointestinal tract).

The story so far

Over the last few years it’s become clear that these co-residents — collectively called the microbiota — are not just free-loaders. They’re critical to our well-being in helping to fight infection by other microrganisms (as we noted in Our Inner Self), they influence our immune system and in the gut they extract the last scraps of nutrients from our diet. So maybe it makes them easier to live with if we keep in mind that we need them every bit as much as they depend on us.

We now know that there are about 2000 different species of bacteria in the human gut (yes, that really is 2,000 different types of bug) and, with all that diversity, it’s not surprising that the total number of genes they carry far exceeds our own complement (by several million to about 20,000). In it’s a small world we noted that obesity causes a switch in the proportions of two major sub-families of bacteria, resulting in a decrease in the number of bug genes. The flip side is that a more diverse bug population (microbiome) is associated with a healthy status. What’s more, shifts of this sort in the microbiota balance can influence cancer development. Even more remarkably, we saw in Hitchhiker Or Driver? That the microbiome may also play a role in the spread of tumours to secondary sites (metastasis).

Time for a deep breath

If all this is going on in the intestines you might well ask “What about the lungs?” — because, and if you didn’t know you might guess, their job of extracting oxygen from the air we inhale means that they are covered with the largest surface area of mucosal tissue in the body. They are literally an open invitation to passing microorganisms — as we all know from the ease with which we pick up infections.

In view of what we know about gut bugs a rather obvious question is “Could the bug community play a role in lung cancer?” It’s a particularly pressing question because not only is lung cancer the major global cause of cancer death but 70% lung cancer patients have bacterial infections and these markedly influence tumour development and patient survival. Tyler Jacks, Chengcheng Jin and colleagues at the Massachusetts Institute of Technology approached this using a mouse model for lung cancer (in which two mutated genes, Kras and P53 drive tumour formation).

In short they found that germ-free mice (or mice treated with antibiotics) were significantly protected from lung cancer in this model system.

How bacteria can drive lung cancer in mice. Left: scheme of a lung with low levels of bacteria and normal levels of immune system cells. Right: increased levels of bacteria accelerate tumour growth by stimulating the release of chemicals from blood cells that in turn activate cells of the immune system to release other effector molecules that promote tumour growth. The mice were genetically altered to promote lung tumour growth (by mutation of the Kras and P53 genes). In more detail the steps are that the bacteria cause macrophages to release interleukins (IL-1 & IL-23) that stick to a sub-set of T cells (γδ T cells): these in turn release factors that drive tumour cell proliferation, including IL-22. From Jin et al. 2019.

As lung tumours grow in this mouse model the total bacterial load increases. This abnormal regulation of the local bug community stimulates white blood cells (T cells present in the lung) to make and release small proteins (cytokines, in particular interleukin 17) that signal to neutrophils and tumour cells to promote growth.

This new finding reveals that cross-talk between the local microbiota and the immune system can drive lung tumour development. The extent of lung tumour growth correlated with the levels of bacteria in the airway but not with those in the intestinal tract — so this is an effect specific to the lung bugs.

Indeed, rather than the players prominent in the intestines (Bs & Fs) that we met in Hitchhiker Or Driver?, the most common members of the lung microbiome are Staphylococcus, Streptococcus and Lactobacillus.

In a final twist Jin & Co. took bacteria from late-stage tumours and inoculated them into the lungs of mice with early tumours that then grew faster.

These experiments have revealed a hitherto unknown role for bacteria in cancer and, of course, the molecular signals identified join the ever-expanding list of potential targets for drug intervention.

References

Jin, C. et al. (2019). Commensal Microbiota Promote Lung Cancer Development via γδ T Cells. Cell 176, 998-1013.e16.

Mosaic Masterpieces

 

A few years ago in one of these pieces I exhorted you to visit the Villa Romana del Casale in the middle of Sicily. What? You still haven’t been?! Shame on you!! It’s even more of a good idea now than it was then.

As we explained in Molecular Mosaics, the villa houses the most extensive set of mosaics anywhere in the world and we were prompted to draw a parallel between their complexity and that of cancer by one of several pieces of research that took small bits of primary and secondary tumours, hit them with the power of DNA sequencing and showed that every region was different. It’s turned out that each individual tumour cell has sequence differences from even its nearest neighbours. So if you look at DNA sequence in cells across a tumour it would indeed be a mosaic.

Beautiful mosaics but …

Wonderful though DNA is, whilst it contains all the information required for life it doesn’t show you how to use it. It’s a bit like an architect’s plan for a new building: immensely detailed as to shape, where the doors and windows are etc., but with no information about the different bits — which is steel or glass, concrete or wood — yet alone how they’re put together to make the final edifice.

DNA is indeed a ‘blueprint’ as it’s so often described but to find out how cells behave we need to know which proteins are being produced at any time by the machinery of the cell as it translates the sequence of DNA bases (A, C, G & T) into the molecules that actually make things happen.

Detecting a killer

So it’s the proteins in a cell that control what the cell can do and indeed define what type of cell it is and it’s worth a moment to look at how modern technology can enable us to pick out individual cells from the 200 or so different types that make up a human being. In principle it’s fairly easy: add antibodies (proteins made by white blood cells that stick to other proteins — antigens) to your sample, chosen to recognise proteins known to be present on cell surfaces. If the antibody has an attached ‘flag’ — e.g., a fluorescent molecule (reviewed in I Know What I Like) you can pick out your cells with the aid of a microscope:

 

Killer T cells in action. The image shows a group of killer T cells (a sub-group of lymphocytes that destroy target cells identified as abnormal, including virus-infected cells and tumour cells) surrounding a cancer cell. DNA is stained blue. The T cells are green (via an antibody that attaches to a surface protein (CD8) that defines this type of cell) and red (small sacs within the cells that deliver ‘the kiss of death’ to kill the cancer cell).

The bigger picture

That’s OK for one type of cell but how can you look at a piece of tissue and ask: “What is the range of cell types present?”  That’s the question Leeat Keren, Michael Angelo and colleagues at Stanford University posed in the context of breast tumours and the stunning answers are to be found in their recent paper in the journal Cell. The methods they used are formidable but, armed with what we’ve just said about detecting one type of lymphocyte (killer T cells), we can break them down into simple steps from which emerge astonishingly complex patterns.

How’s it done?

They used a panel of antibodies that could pick out 36 different cell types and added them to slices of tumours. Rather than using fluorescent labels their antibodies carried ‘mass tags’. When a beam of charged particles is fired at the sample (so that it rasters across the target) cells are nebulized into single-cell droplets. The location of fragments released carrying the tags can be pinpointed and they are analysed by mass spectrometry to identify the antibody. From this comes an image of protein expression (i.e. cell type), each being given a false colour (or pseudo colour) to show up the distribution.

Cellular architecture of tumour samples. Each panel is a section of a breast tumour: each is from a different patient. Individual cell types picked out as described above. From Keren et al. 2018.

Not just a pretty picture

You might think this is just the tile pattern you’ve been after for your kitchen — not least because the most striking feature is that no two pieces are the same. The biology behind this variation is that across patients there were large differences in both the variety (type) and number of immune cells, notwithstanding the fact that all had the same type of tumour — triple-negative breast cancer.

However, even from this extraordinary variation it was possible to tease out some trends. Thus, for example, some tumours had high numbers of macrophages (25%) but low numbers of killer cells (1%), with B cells (11%), CD4+ (15%), CD8+ (19%) and regulatory T cells (1%) falling in between. An important point is that these trends relate to patient survival. The variation in patterns also hints at how different cells of the immune system are drawn to the site of a tumour and hence how they might cooperate in mounting a defence against cancer progression. Another notable finding was that proteins known to be important in controlling the immune response (e.g., PD-L1) can appear in different cell types. For example, in tumour cells themselves in some patients but in immune cells in others.

It’s amazing science, complete with pretty pictures — but it should help in categorizing patients and in the rational design of therapies.

References

Keren, L. et al. (2018). A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging. Cell 174, P1373-P1387.E19.

Sticky Cancer Genes

 

In the previous blog I talked about Breath Biopsy — a new method that aims to detect cancers from breath samples. I noted that it could end up complementing liquid biopsies — samples of tumour cell DNA pulled out of a teaspoon of blood — both being, as near as makes no difference, non-invasive tests. Just to show that there’s no limit to the ingenuity of scientists, yet another approach to the detection problem has just been published — this from Matt Trau and his wonderful team at The University of Queensland.

This new method, like the liquid biopsy, detects DNA but, rather than the sequence of bases, it identifies an epigenetic profile — that is, the pattern of chemical tags (methyl groups) attached to bases. As we noted in Cancer GPS? cancer cells often have abnormal DNA methylation patterns — excess methylation (hypermethylation) in some regions, reduced methylation in others. Methylation acts as a kind of ‘fine tuner’, regulating whether genes are switched on or off. In the methylation landscape of cancer cells there is an overall loss of methylation but there’s an increase in regions that regulate the expression of critical genes. This shows up as clusters of methylated cytosine bases.

Rather helpfully, a little while ago in Desperately SEEKing … we talked about epigenetics and included a scheme showing how differences in methylation clusters can identify normal cells and a variety of cancers and how these were analysed in the computer program CancerLocator.

The Trau paper has an even better scheme showing how the patterns of DNA decoration differ between normal and cancer cells and how this ‘methylscape’ (methylation landscape) affects the physical behaviour of DNA.

How epigenetic changes affect DNA. Scheme shows methylation (left: addition of a methyl group to a cytosine base in DNA) in the process of epigenetic reprogramming in cancer cells. This change in the methylation landscape affects the solubility of DNA and its adsorption by gold (from Sina et al. 2018).

Critically, normal and cancer epigenomes differ in stickiness — affinity — for metal surfaces, in particular for gold. In a clever ploy this work incorporated a colour change as indicator. We don’t need to bother with the details — and the result is easy to describe. DNA, extracted from a small blood sample, is added to water containing tiny gold nanoparticles. The colour indicator makes the water pink. If the DNA is from cancer cells the water retains its original colour. If it’s normal DNA from healthy cells the different binding properties turns the water blue.

By this test the Brisbane group have been able to identify DNA from breast, prostate and colorectal cancers as well as from lymphomas.

So effective is this method that it can detect circulating free DNA from tumour cells within 10 minutes of taking a blood sample.

The aim of the game — and the reason why so much effort is being expended — is to detect cancers much earlier than current methods (mammography, etc.) can manage. The idea is that if we can do this not weeks or months but perhaps years earlier, at that stage cancers may be much more susceptible to drug treatments. Trau’s paper notes that their test is sensitive enough to detect very low levels of cancer DNA — not the same thing as early detection but suggestive none the less.

So there are now at least three non-invasive tests for cancer: liquid biopsy, Breath Biopsy and the Queensland group’s Methylscape, and in the context of epigenetics we should also bear in mind the CancerLocator analysis programme.

Matt Trau acknowledges, speaking of Methylscape, that “We certainly don’t know yet whether it’s the holy grail for all cancer diagnostics, but it looks really interesting as an incredibly simple universal marker for cancer …” We know already that liquid biopsies can give useful information about patient response to treatment but it will be a while before we can determine how far back any of these methods can push the detection frontier. In the meantime it would be surprising if these tests were not being applied to age-grouped sets of normal individuals — because one would expect the frequency of cancer indication to be lower in younger people.

From a scientific point of view it would be exciting if a significant proportion of ‘positives’ was detected in, say, 20 to 30 year olds. Such a result would, however, raise some very tricky questions in terms of what, at the moment, should be done with those findings.

Reference

Abu Ali Ibn Sina, Laura G. Carrascosa, Ziyu Liang, Yadveer S. Grewal, Andri Wardiana, Muhammad J. A. Shiddiky, Robert A. Gardiner, Hemamali Samaratunga, Maher K. Gandhi, Rodney J. Scott, Darren Korbie & Matt Trau (2018). Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker. Nature Communications 9, Article number: 4915.

Born On Wings

 

Alexander Porfiryevich Borodin is a name that will perhaps be familiar only to musical folk of a fairly dedicated kind. Which is a shame because he wrote some wonderful music particularly in his symphonies, in the magical portrait of the steppes of Central Asia and in his opera Prince Igor, albeit not finishing the latter. But Borodin was more than just a gifted composer for he started life as an illegitimate child, qualified as a doctor in Saint Petersburg and became a Professor of Chemistry at the Imperial Medical-Surgical Academy in that city. He carried out some very significant chemical research – he’s even got a reaction named after him – whilst, as a hobby, becoming a sufficiently outstanding composer to be one of The Mighty Handful. Along the way he founded the School of Medicine for Women in Saint Petersburg, the first Russian medical institute for women.

Advanced chemistry

With that background we can be sure that Borodin would have been thrilled to note the recent headlines about the trial of a breathalyser test for cancers. It’s being run by my colleague Rebecca Fitzgerald of the Cancer Research UK Cambridge Centre (and of tea bag fame: see Open Wide for Pasty’s Throat), initially for people suspected to have oesophageal or stomach cancers but in time to be extended to other cancers. What would have excited the chemist in Borodin is that the test collects airborne molecules from the breath and uses the most advanced modern chemistry to analyse them (the details don’t matter but, for the record, the critical method is called Field Asymmetric Ion Mobility Spectroscopy (FAIMS) which distinguishes molecules by how fast they move when driven through a gas by an electric field).

What’s new?

At first pass it may sound fanciful to think of detecting cancer on the breath but perhaps it shouldn’t. After all, we’re familiar with breathalysers that detect alcohol levels and, more generally, we all know that ‘bad breath’ isn’t a good sign. For example, the smell of acetone on the breath can arise from type I diabetes, when the body increases its use of fat due to low insulin levels. My old chemistry teacher was known throughout the school as ‘Fruity’ — the word he used with relish to describe the scent of ketones (acetone is the smallest member of the ketones).

The general point is that molecules released from cells can find their way into the lungs and emerge in the breath and now they can be identifed to find signatures indicative of disease.

The detection of breath-born chemicals can inform diagnosis and treatment of disease. From ebook “Breath Biopsy: The Complete Guide” by Owlstone Medical Ltd.

Pioneering this approach is a company called Owlstone Medical whose Breath Biopsy analytical platform carries out the spectroscopy of airborne molecules (volatile organic compounds). The idea is that someone exhales into a mask and chemicals born on the breath are collected by a cartridge for subsequent analysis. More than 1000 different compounds can be identified by this state-of-the-art technology and for cancer detection these may include substances released by tumour cells and also those emanating from host cells that have been drawn into the tumour microenvironment.

Followers of this blog will know of my enthusiasm for cancer early detection, in particular the liquid biopsy method that permits the isolation of tumour DNA from small blood samples. The breathalyzer system is a different approach to the same problem — and it may be that, in the end, both will have useful roles to play. I should add that my publicizing Owlstone Medical is entirely on account of the apparent potential of their system for cancer screening. Although the company is based in Cambridge, I have no connection with them. I rather wish I had.

If Borodin was here to comment he might wryly observe that in his opera the breaths launched by the enslaved captives when they start to sing ‘Born on wings …’ carried only grief and sorrow but with Breath Biopsy it may be that bad news enwraps good — if it carries a warning of cancers or other diseases sufficiently early that they may be stopped in their tracks.

I Know What I Like

 

I guess most of us at some time or other will have stood gazing at a painting for a while before muttering ‘Wow, that’s awesome’ or words to that effect if we’re not into the modern argot. Some combination of subject, style and colour has turned our crank and left us thinking we wouldn’t mind having that on our kitchen wall.

Given the thousands of years of man’s daubing and the zillions of forms that have appeared from pre-historic cave paintings through Eastern painting, the Italian Renaissance, Impressionism, Dadaism and the rest to Pop Art, it’s amazing that everyone isn’t a fanatic for one sort or another. The sane might say the field’s given itself a bad name by passing off tins of baked beans, stuff thrown at a canvas and unmade beds as ‘art’ but, even so, it seems odd that it remains a minority obsession.

Can science help?

Science is wonderful, as we all know, but the notion that it might arouse the collective artistic lust seems fanciful. Nevertheless, unnoticed by practically everyone, our vast smorgasbord of smears has been surreptitiously joined over the last 30 years by a new form: an ever-expanding avalanche of pics created by biologists trying to pin down how animals work at the molecular level. The crucial technical development has been the application of fluorescence in the life sciences: flags that glow when you shine light on them and can be stuck on to molecules to track what goes on in cells and tissues. The pioneer of this field was Roger Tsien who died, aged 64, in 2016.

Because this has totally transformed cell biology we’ve run into lots of brilliant examples in these pages — recently in Shifting the Genetic Furniture, in Caveat Emptor and John Sulston: Biologist, Geneticist and Guardian of our Heritage and in the use of red and green tags for picking out individual types of proteins that mark mini-cells within cells in Lorenzo’s Oil for Nervous Breakdowns.

To mark the New Year this piece looks at science from a different angle by focussing not on the scientific story but on the beauty that has become a by-product of this pursuit of knowledge.

Step this way: entrance free

So let’s take a stroll through our science gallery and gaze at just a few, randomly selected works of art.

  1. Cells grown in culture:

This was one of the first experiments in my laboratory using fluorescently labelled antibodies, carried out by a student, Emily Hayes, so long ago that she now has a Ph.D., a husband and two children. The cells are endothelial cells (that line blood vessels). Blue: nuclei; green: F-actin; red: Von Willebrand factor, a protein marker for endothelium.

 

  1. Two very recent images taken by my colleague Roderik Kortlever of a senescent mouse fibroblast and of mouse breast tissue:

 

 

 

 

 

 

3. Waves of calcium in firing neurons:

One of my fondest memories is helping to do the first experiment that measured the level of calcium within a cell, carried out with my colleague the late Roger Tsien and two other friends. I only grew the cells: Roger had designed and made the molecule, quin2. We didn’t know it at the time but Roger’s wonder molecule was the first of many intracellular ‘reporters.’ Roger shared the 2008 Nobel Prize in Chemistry for his discovery and development of the green fluorescent protein with organic chemist Osamu Shimomura and neurobiologist Martin Chalfie.

This wonderful video of a descendant of quin2 in nerve cells was made in Dr. Sakaguchi’s lab at Iowa State University.

 

4. Calcium wave flooding a fertilized egg: Taro Kaneuchi and colleagues at the Tokyo Metropolitan University:

Click for a time-lapse movie of an egg cell that has been artificially stimulated to show the kind of calcium change that happens at fertilization. In this time-lapse movie the calcium level reaches a maximum signal intensity after about 30 min before gradually decreasing to the basal level.

 

5. The restless cell (1):

This movie shows how protein filaments in cells can continuously break down and reform – called treadmilling. Visualised in HeLa cells using a green fluorescent protein that sticks to microtubules (tubular polymers made up of the protein tubulin) by HAMAMATSU PHOTONICS.

 

6. The restless cell (2):

This movie shows how mitochondria (organelles within the cell) are continuously changing shape and moving within the cell’s interior (cytosol). Red marks the mitochondria; green DNA within the nucleus. HAMAMATSU PHOTONICS.

 

7. Cell division:

Pig kidney cells undergoing mitosis. Red marks DNA (nucleus); green is tubulin: HAMAMATSU PHOTONICS.

 

8. DNA portrait of Sir John Sulston by Marc Quinn commissioned by the National Portrait Gallery:This image looks a bit drab in the present context but in some ways it’s the most dramatic of all. John Sulston shared the 2002 Nobel Prize in Physiology or Medicine with Sydney Brenner and Robert Horvitz for working out the cell lineage of the roundworm Caenorhabditis elegans (i.e. how it develops from a single, fertilized egg to an adult). He went on to sequence the entire DNA of C. elegans. Published in 1998, it was the first complete genome sequence of an animal — an important proof-of-principle for the Human Genome Project that followed and for which Sulston directed the British contribution at the Sanger Centre in Cambridgeshire, England. The project was completed in 2003.

The portrait shows colonies of bacteria in a jelly that, together, carry all Sulston’s DNA. This represents DNA cloning in which DNA fragments, taken up by bacteria after insertion into a circular piece of DNA (a plasmid), are multiplied to give many identical copies for sequencing.

 

9. “Brainbow” mice by Tamily Weissman at Harvard University:

The science behind this astonishing image builds on the work of Roger Tsien. Mice are genetically engineered to carry three different fluorescent proteins corresponding to the primary colours red, yellow and blue. Within each cell recombination occurs randomly, giving rise to different colours. The principle of mixing primary colours is the same as used in colour televisions.  In this view individual neurons in the brain (specifically a layer of the hippocampus) project their dendrites into the outer layer. Other magnificent pictures can be seen in the Cell Picture Show.

It’s certainly science – but is it art?

A few years ago the Fitzwilliam Museum in Cambridge staged Vermeer’s Women, an exhibition of key works by Johannes Vermeer and over thirty other masterpieces from the Dutch ‘Golden Age’. I tried the experiment of standing in the middle of each room and picking out the one painting that, from a distance, most caught my amateur eye. Funny thing was: not one turned out to be by the eponymous star of the show! Wondrous though Vermeer’s paintings were, the ones that really took my fancy were by Pieter de Hooch, Samuel van Hoogstraten and Nicolaes Maes, guys I’d never heard of.

Which made the point that you don’t need to be a big cheese to make a splash and that in the new Dutch Republic of the 17th century, the most prosperous nation in Europe, there was enough money to keep a small army of splodgers in palettes and paint. Skillful and incredibly patient though these chaps were, they simply used the tools available to paint what they saw in the world before them — as for the most part have artists down the ages.

But hang on! Isn’t that what we’ve just been on about? Scientists applying enormous skill and patience in using the tools they’ve developed to visualize life — to image what Nature lays before them. So the only difference between the considerable army of biological scientists around the world making a new art form and the Old Masters is that the newcomers are unveiling life — as opposed to the immortalizing a rather dopy-looking aristocrat learning to play the virginal or some-such.

Controversial?

Not really. Let’s leave the last word to Roger Tsien. In our final picture there are eight bacterial colonies each expressing a different colour of fluorescent protein arranged to grow as a San Diego beach scene in a Petri dish. It became the logo of Roger’s laboratory.

Shifting the Genetic Furniture

 

Readers of these pages will know very well that cells are packets of magic. Of course, we often describe them in the simplest terms: ‘Sacs of gooey stuff with lots of molecules floating around.’ And it’s true that we know a lot about the protein pathways that capture energy from the food we eat and about the machinery that duplicates genetic material, makes new proteins and sustains life. Even so, although we’ve worked out much molecular detail, we have scarcely a clue about how ‘stuff’ in cells is organised. How do the tens of thousands of different types of proteins find their places in the seemingly chaotic jumble of a cell so that they can do their job? If that remains a mystery there’s an even more perplexing one in the form of the nucleus. That’s a smaller sac (i.e. a compartment surrounded by a membrane) that is home to most of our genetic material — i.e. DNA.

Sizing up the problem

It’s easy to see why evolution came up with the idea of a separate enclosure for DNA which only has to do two things: reproduce itself and enable regions of its four base code to be transcribed into molecules that can cross the nuclear membrane to be translated into proteins in the body of the cell. But here’s the puzzle. The nucleus is very small and there’s an awful lot of DNA — over 3,000 million bases in each of the two strands of human DNA (and, of course, two complete sets of chromosomes go to make up the human genome) — so 2 metres of it in every cell. A rather pointless exercise, unless you go in for pub quizzes, is to work out the length of all your DNA if you put it together in a single string. 1013 cells (i.e. 1 followed by 13 zeros) in your body: 2 metres per cell. Answer: your DNA would stretch to the sun and back 67 times.

Mmm. More relevantly, the nucleus of a cell is typically about 6 micrometres (µm) in diameter — that’s six millionths of a metre (6/1,000,000 metre), into which our 2 metres must squeeze.

Time for some serious packing to be done but it’s not just a matter of stuffing it in any old how and sitting on the lid. As we’ve just noted, every time cells divide all the DNA has to be replicated and regions (i.e. genes) are continually being “read” to make proteins. So the machinery in the nucleus has to be able to get at specific regions of DNA and disentangle them sufficiently for code reading. Part of evolution’s solution to these problems has been to add proteins called histones to DNA (the term chromosome refers to DNA together with histone packaging proteins and other proteins). To understand how this leads to “more being less”, consider DNA as a length of cotton. If you just scrunch the cotton up into a ball you get a tangled mess. But if you use cotton reels (aka histones — two or three hundred million per cell), you can reduce the great length to smaller, more organized blocks — which is just as well because they’re all that stands between life and a tangled mess.

Thinking of histones as cotton reels helps a bit in thinking about how the nucleus achieves the seemingly impossible but the fact of the matter is that we have no real idea about how DNA is unravelling is controlled so that the two strands can be unzipped and replicated, yet alone the way in which starting points for reading genes are found by proteins.

Undeterred by our profound ignorance Haifeng Wang and colleagues at Stanford University have just done something really amazing. They came up with a way of moving regions of DNA from the jumble of the nuclear interior to the membrane and they showed that this can change the activity of genes. They used CRISPR (that we described in Re-writing the Manual of Life) to insert a short piece of DNA next to a chosen gene. The insert was tagged with a protein designed to attach to a hormone that also binds to a protein (called emerin) that sits in the nuclear membrane. So the idea was that when the hormone is added to cells it can hook on to the DNA tag and, by attaching to emerin, can drag the chosen gene to the membrane. The coupling agent is a plant hormone (abscisic acid) although it also occurs in other species, including humans. Wang & Co christened their method CRISPR-GO for CRISPR-Genome Organizer.

Tagging a DNA insert with a protein so that a coupling molecule can pull a region of DNA to a protein in the membrane of the nucleus. From Wang et al., 2018.

Repositioning regions of DNA in the nucleus. DNA is labeled blue which defines the shape of the nucleus. Red dots are specific genes before (left) and after (right) adding the coupling agent. From Pennisi 2018.

How did CRISPR-GO go?

Astonishingly well. Not only could it shift tagged DNA from the interior to the membrane of the nucleus but the rearrangements could change the way cells behaved. Depending on which regions were moved and where to, cells grew more slowly or more rapidly.

So this is a really remarkable technical feat — but it’s not just molecular pyrotechnics for fun. It looks as though this approach may offer at long last a way of dissecting how cells go about getting a controlled response out of the mind-boggling complexity that is their genetic material.

References

Wang, H. et al. (2018). CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell 175, 1405-1417.

Pennisi, E. (2018). Moving DNA to a different part of the nucleus can change how it works. Science Oct. 11th.

Food Fix For Pharma Failure

 

If you held a global quiz, Question: “Which biological molecules can you name?” I guess, setting aside ‘DNA‘, the top two would be insulin and glucose. Why might that be? Well, the World Health Organization reckons diabetes is the seventh leading cause of death in the world. The number of people with diabetes has quadrupled in the last 30 years to over 420 million and, together with high levels of blood glucose (sugar), it kills nearly four million a year.

There are two forms of diabetes: in both the level of glucose in the blood is too high. That’s normally regulated by the hormone insulin, made in the pancreas. In Type 1 diabetes insulin isn’t made at all. In Type 2 insulin is made but doesn’t work properly.

When insulin is released into the bloodstream it can ‘talk’ to cells by binding to protein receptors that span cell membranes. Insulin sticks to the outside, the receptor changes shape and that switches on signalling pathways inside the cell. One of these causes transporter molecules to move into the cell membrane so that they can carry glucose from the blood into the cell. When insulin doesn’t work it is this circuit that’s disrupted.

Insulin signalling. Insulin binds to its receptor which transmits a signal across the cell membrane, leading to the activation of the enzyme PIK3. This leads indirectly to the movement of glucose transporter proteins to the cell membrane and influx of glucose.

So the key thing is that, under normal conditions, when the level of blood glucose rises (after eating) insulin is released from the pancreas. Its action (via insulin receptors on target tissues e.g., liver, muscle and fat) promotes glucose uptake and restores normal blood glucose levels. In diabetes, one way or another, this control is compromised.

Global expansion

Across most of the world the incidence of diabetes, obesity and cancer are rising in parallel. In the developed world most people are aware of the link between diabetes and weight: about 90% of adults with diabetes are overweight or obese. Over 2 billion adults (about one third of the world population) are overweight and nearly one third of these (31%) are obese — more than the number who are underweight. The cause and effect here is that obesity promotes long-term inflammation and insulin resistance — leading to Type 2 diabetes.

Including cancer

The first person who seems to have spotted a possible connection between diabetes and cancer was the 19th-century French surgeon Theodore Tuffier. He was a pioneer of lung and heart surgery and of spinal anaesthesia and he’s also a footnote in the history of art by virtue of having once owned A Young Girl Reading, one of the more famous oil paintings produced by the prolific 18th-century artist Jean-Honoré Fragonard (if you want to see it head for the National Gallery of Art in Washington DC). Tuffier noticed that having type 2 diabetes increased the chances of patients getting some forms of cancer and pondered whether there was a relationship between diabetes and cancer.

It was a good question then but it’s an even better one now when this duo have become dominant causes of morbidity and mortality worldwide.

We now know that being overweight increases the risk of a wide range of cancers including two of the most common types — breast and bowel cancers. Unsurprisingly, the evidence is also clear that diabetes (primarily type 2) is associated with increased risk for some cancers (liver, pancreas, endometrium, colon and rectum, breast, bladder).

With all this inter-connecting it’s perhaps not surprising that the pathway by which insulin regulates glucose also talks to signalling cascades involved in cell survival, growth and proliferation — in other words, potential cancer initiators. The central player in all this is a protein called PIK3 (it’s an enzyme that adds phosphate groups (so it’s a ‘kinase’) to a lipid called phosphatidylinositol bisphosphate, an oily, water-soluble component of the plasma membrane). It’s turned out that PIK3 is one of the most commonly mutated genes in human cancers — e.g., PIK3 mutations occur in 25–40% of all human breast cancers.

Signalling pathways switched on by mutant PIK3. A critical upshot is the activation of cell survival and growth that leads to cancer.

Accordingly, much effort has gone into producing drugs to block the action of PIK3 (or other steps in this signal pathway). The problem is that these have worked as cancer treatments either very variably or not at all.

The difficulty arises from the inter-connectivity of signalling that we’ve just described: a drug blocking insulin signalling causes the liver to release glucose and prevents muscle and fats cells from taking up glucose. Result: blood sugar levels rise (hyperglycaemia). This effect is usually transient as the pancreas makes more insulin that restores normal glucose levels.

Blockade of mutant PIK3 by an inhibitor. This blocks the route to cancer but glucose levels rise in the circulation (hyperglycaemia) promoting the release of insulin (top). Insulin can now signal through the normal pathway (bottom), overcoming the effect of the anti-cancer drug. Note that the cell has two copies of the PIK3 gene/protein, one of which is mutated, the other remaining normal.

Is our journey really necessary?

By now you might be wondering whether there is anything that makes grappling with insulin signaling worth the bother. Well, there is — and here it is. It’s a recent piece of work by Benjamin Hopkins, Lewis Cantley and colleagues at Weill Cornell Medicine, New York who looked at ways of getting round the insulin feedback response so that the effect of PIK3 inhibitors could be boosted.

Sketch showing the effect of diet on the potency of an anti-cancer drug in mice. The red line represents normal tumour growth. The black line shows the effect of PIK3 blockade when the mice are on a ketogenic diet: tumour growth is suppressed. On a normal diet the drug alone has only a slight effect on tumour growth. Similar results were obtained in a variety of model tumours (Hopkins et al., 2018).

They first showed that, in a range of model tumours in mice, insulin feedback caused by blockade of PIK3 was sufficient to switch on signalling even in the continued presence of anti-PIK3 drugs. The really brilliant result was that changing the diet of the mice could offset this effect. Switching the mice to a high-fat, adequate-protein, low-carbohydrate (sugar) diet essentially stopped the growth of tumours driven by mutant PIK3 treated with PIK3 blockers. This is a ketogenic (or keto) diet, the idea being to deplete the store of glucose in the liver and hence limit the rise in blood glucose following PIK3 blockade.

Giving the mice insulin after the drug drastically reduces the effect of the PIK3 inhibitor, supporting the idea that that a keto diet improves responses to PIK3 inhibitors by reducing blood insulin and hence its capacity to switch on signalling in tumour cells.

A few weeks prior to the publication of the PIK3 results another piece of work showed that adding the amino acid histidine to the diet of mice can increase the effectiveness of the drug methotrexate against leukemia. Methotrexate was one of the first anti-cancer agents to be made and has been in use for 70 years.

These are really remarkable results — as far as I know the first time diet has been shown to influence the efficacy of anti-cancer drugs. It doesn’t mean that all tumours with mutations in PIK3 have suddenly become curable or that the long-serving methotrexate is going to turn out to be a panacea after all — but it does suggest a way of improving the treatment of many types of tumour.

References

Hopkins, B.D. et al. (2018). Suppression of insulin feedback enhances the efficacy of PI3K inhibitors. Nature 560, 499-503.

Kanarek, N. et al. (2018). Histidine catabolism is a major determinant of methotrexate sensitivity. Nature 559, 632–636.