Mushrooming Secret Army

 

We have in these pages talked quite a bit about our ‘secret army’ — the bugs that share our body to the extent that bacteria outnumber us on a cell-to-cell basis by at least three to one. As we noted in Secret Army: More Manoeuvres Revealed, bacteria are just one part of what is collectively called the microbiota’ but with over 2000 different species and a total gene pool hundreds of times bigger than our own 20,000 or so, they are by far the biggest. And it’s gradually become clear that they are not with us just because our bodies are warm, damp and comfortable but they help us get the most out of our food and they’re important in the working of our immune system.

Bacteria and cancer

Most critically, in the present context, we now know that shifts in proportions of species in the microbiome can influence cancer development and perhaps even the spread of tumour cells around the body.

Small fry

Important though they are, bacteria aren’t the only members of the microbiome — which includes fungi, viruses and various single-celled parasites (protozoa). Today’s story is about fungi, a group of microorganisms familiar to gardeners world-wide, that includes yeasts and molds, as well as the more familiar mushrooms. There’s estimated to be several million species of fungi, although only about 120,000 have been described. Some we can eat, some can kill us and, of course, there’s magic mushrooms.

With all this diversity you might wonder whether any fungi have elbowed their way into us to share the delights of the human body alongside bacterial microbes. Of course they have: most people will have heard of candidiasis — a fungal infection caused by Candida yeasts that belong to the genus Candida. Candida normally finds its niche in places like the mouth (giving the condition called thrush), gut, vagina and on the skin and usually doesn’t give us any trouble. But, truth to tell, we’ve known very little about fungi in us until recently when the power of DNA sequencing has started to be applied to the topic. This has confirmed that we do carry lots of fungi around with us, albeit that they are only a tiny fraction of the microbial community (somewhat less than 0.1%).

New actor in the cancer cast

This fungal force of microbes is known as the mycobiome (as distinct from the microbiome) and, in contrast to bacteria, there is no evidence that it has a role in cancer. Until, that is, the recent publication from New York University School of Medicine by Berk Aykut, George Miller and friends showing that fungi travel from the gut to the pancreas where a particular species can actually give cancer a helping hand. The cancer in question is pancreatic ductal adenocarcinoma (PDA) that has a particularly dismal prognosis.How a fungus can drive cancer. The scheme represents a tumour in the pancreas changing the make up of the adjacent fungal community and how a protein in the blood called mannose binding lectin (MBL) can attach to the outer surface of a fungal cell. When this happens MBL changes shape so it can then stick to another protein (C3) which in turn activates a relay of proteins called the complement cascade. One upshot of this can be to promote tumour growth. From Dambuza and Brown 2019.

How did they do it?

Aykut et al. first used DNA sequencing to look for fungus-specific sequences in the pancreas of humans with PDA and in mouse models of PDA, They’d previously shown that the bacterial load goes up by about 1000-fold in tumours compared with healthy tissue and, lo and behold, they found a similar increase in fungi. Next they tagged strains of fungus with a fluorescent label and showed that the cells could migrate from the gut to the pancreas of mice in under 30 minutes.

They then tracked down a protein called mannose binding lectin (MBL) expression of which is associated with poor survival in human PDA patients. MBL is a ‘serum protein’, meaning that it floats around in blood. This led to the discovery that MBL can bind to the surface of fungal cells and when it does so changes shape to permit activation of a relay of signal proteins called the complement system. This ‘complement cascade’ is part of our immune system, enhancing the capacity of antibodies and phagocytic cells to clear microbes from the circulation.

Jules Bordet was the chap who first showed that something in normal blood plasma could help to kill off bacteria back at the end of the 19th century and, as such, deserves to be better remembered as a famous Belgian.

The complement system is pretty amazing because, whilst it can trigger an immune response against invading pathogens, it can also switch on inflammatory pathways that help cells grow and move around — in other words, give a helping hand to tumours.

Fungible?

I met this word for the first time a few days ago, courtesy of the journalist and author Ann Treneman. You’d think that no piece on fungi would be complete without it but it turns out to have nothing to do with mushrooms: it just means interchangeable or switchable. But hang on! We can squeeze it in by asking a very relevant question: are pancreatic fungi fungible in terms of their capacity to promote cancer? Aykut et al. did just that and the answer was ‘no they’re not.’ One species seems to be particularly abundant in PDA: the genus Malassezia. This was true for both mouse and human tumours and perhaps that shouldn’t surprise us as Malassezia is the most abundant fungal species in mammalian skin, accounting for more than 80% of our skin mycobiome. So it’s Malassezia not other species (e.g., Candida) that has the power to drive cancer.

Spores of the yeast Malassezia

Fungal footnote

In a final exciting experiment Aykut et al. showed that antifungal drugs halted PDA progression in mice and improved the ability of chemotherapy to shrink the tumour. This obviously raises the notion that if we can find ways of shifting the balance of fungal communities or interfering with the link to the complement cascade we might have a completely new line on desperately needed therapies for this disease.

References

Aykut, B. et al., (2019). The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL. Nature 574, 264–267.

Dambuza, I.M. and Brown, G.D. (2019). Fungi accelerate pancreatic cancer. Nature 574, 184-185.

The Power of Flower

 

We know we don’t ‘understand cancer’ — for if we did we would at least be well on the way to preventing the ten million annual deaths from these diseases and perhaps even stymieing their appearance in the first place. But at least, after many years of toil by thousands of curious souls, we might feel brave enough to describe the key steps by which it comes about.

Here goes!

Our genetic material, DNA, carries a code of four different units (bases) that enables cells to make twenty-thousand or so different types of proteins. Collectively these make cells — and hence us — ‘work’. An indicator of protein power is that we grow from single, fertilized cells to adults with 50 trillion cells. That phenomenal expansion involves, of course, cells growing and dividing to make more of themselves — and, along the way, a bit of cell death too. The fact that there are nearly eight billion people on planet earth testifies to the staggering precision with which these proteins act.

Nobody’s perfect

As sports fans will know, the most successful captain in the history of Australian rugby, John Eales, was nicknamed ‘Nobody’ because ‘Nobody’s perfect’. Well, you might care to debate the infallibility of your sporting heroes but when it comes to their molecular machinery, wondrous though it is, perfect it is not.

Evidence: from the teeming eight billion there emerges every year 18 million new cancer cases (that’s about one in every 444). And cancers are, of course, abnormal cell growth: cells growing faster than they should or growing at the wrong time or in the wrong place — any of which means that some of the masterful proteins have suffered a bit of a malfunction, as the computer geeks might say.

How can that happen?

Abnormal protein activity arises from changes in DNA (mutations) that corrupt the normal code to produce proteins of greater or lesser activity or even completely novel proteins.

These mutations may be great or small: changes in just one base or seismic fragmentation of entire chromosomes. But the key upshot is that the cell grows abnormally because regulatory proteins within the cell have altered activity. Mutations can also affect how the cell ‘talks’ to the outside world, that is, the chemical signals it releases to, for example, block immune system killing of cancer cells.

Clear so far?

Mutations can change how cells proliferate, setting them free of normal controls and launching their career as tumour cells and, in addition, they can influence the cell’s environment in favour of unrestricted growth.

The latter tells us that cancer cells cooperate with other types of cell to advance their cause but now comes a remarkable discovery of a hitherto unsuspected type of cellular collaboration. It’s from Esha Madan, Eduardo Moreno and colleagues from Lisbon, Arkansas, St. Louis, Indianapolis, Omaha, Dartmouth College, Zurich and Sapporo who followed up a long-known effect in fruit flies (Drosophila) whereby the cells can self-select for fitness to survive.

Notwithstanding the fact that flies do it, the idea of a kind of ‘cell fitness test’ is novel as far as human cells go — but it shouldn’t really surprise us, not least because our immune system (the adaptive immune system) features much cooperation between different types of cell to bring about the detection and removal of foreign or damaged cells.

Blooming science

So it’s been known for over forty years that Drosophila carries out cell selection based on a ‘fitness fingerprint’ that enables it to prevent developmental errors and to replace old tissues with new. However, it took until 2009 before the critical protein was discovered and, because mutant forms of this protein gave rise to abnormally shaped nerve cells that looked like bunches of flowers, Chi-Kuang Yao and colleagues called the gene flower‘.

Cells can make different versions of flower proteins (by alternative splicing of the gene) the critical ones being termed ‘winner’ and ‘loser’ because when cells carrying winner come into contact with cells bearing loser the latter die and the winners, well, they win by dividing and filling up the space created by the death of losers.

The effect is so dramatic that Madan and colleagues were able to make some stunning movies of the switch in cell populations that occured when they grew human breast cancer cells engineered to express different version of flower tagged with red or green fluorescent labels.

Shift in cell populations caused by two types of flower proteins. 

Above are images at time zero and 24 h later of co-cultures of cells expressing  green and red proteins (losers and winners). From Madan et al. 2019.

Click here to see the movie on the Nature website.

Winner takes almost all

The video shows high-resolution live cell imaging over a 24 hour period compressed into a few seconds. Cells expressing the green protein (hFwe1 (GFP)) were co-cultured with red cells (hFwe2 (RFP)). Greens are losers, reds winners. As the movie progresses you can see the cell population shifting from mainly green to almost entirely red, as the first and last frames (above) show.

How does flower power work?

Flower proteins form channels across the outer membrane of the cell that permit calcium flow, and it was abnormal calcium signalling that caused flowers to bloom in Drosophila nerves. It would be reasonable to assume that changes in calcium levels are behind the effects of flower on cancer cells. Reasonable but wrong, for Madan & Co were able to rule out this explanation. At the moment we’re left with the rather vague idea that flower proteins mediate competitive interactions between cells and these determine whether cells thrive and proliferate or wither and die.

Does this really happen in human cancers?

Madan and colleagues looked at malignant and benign human tumours and found that there was more ‘winner’ flower protein in the former than the latter and that ‘loser’ levels were higher in normal cells next to a tumour than further away. Both consistent with the notion that tumour cells express winner and this induces loser in nearby normal cells leading to their death. What’s more they reproduced this effect in mice by transplanting human breast cancer cells expressing winner.

So there we are! After all this time a variant on how cancer cells can manipulate their surroundings to promote the development of tumours. Remarkable though this finding is, in a way that is familiar it’s just the beginning of this story. We don’t know how flower proteins work in giving cancers a helping hand and we don’t know how effective they are. Until we answer those questions it would be premature to try to come up with therapies to block their effect.

But it is a moment to sit back and reflect on the astonishing complexity of living organisms and their continuing capacity to surprise.

Reference

Madan, E. et al. (2019). Flower isoforms promote competitive growth in cancer. Nature 572, 260-264.

Yao, C-K., et al., (2009). A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis. Cell 138, 947–960.

Breaking Up Is Hard To Do

 

Thus Neil Sedaka, the American pop songster back in the 60s. He was crooning about hearts of course but since then we’ve discovered that for our genetic hearts — our DNA — breaking up is not that tough and indeed it’s quite common.

A moving picture worth a thousand words

When I’m trying to explain cancer to non-scientists I often begin by showing a short movie of a cell in the final stages of dividing to form two identical daughter cells. This is the process called mitosis and the end-game is the exciting bit because the cell’s genetic material, its DNA, has been duplicated and the two identical sets of chromosomes are lined up in the middle of the cell. There ensues a mighty tug-of-war as cables (strands of proteins) are attached to the chromosomes to rip them apart, providing a separate genome for each new cell when, shortly after, the parent cell splits into two. When viewed as a speeded-up movie it’s incredibly dramatic and violent — which is why I show it because it’s easy to see how things could go wrong to create broken chromosomes or an unequal division of chromosomes (aneuploidy). It’s the flip side if you like to the single base changes (mutations) — the smallest damage DNA can suffer — that are a common feature of cancers.

In Heir of the Dog we showed pictures of normal and cancerous chromosomes that had been tagged with coloured markers to illustrate the quite staggering extent of “chromosome shuffling” that can occur.

Nothing new there

We’ve known about aneuploidy for a long time. Over 20 years ago Bert Vogelstein and his colleagues showed that the cells in most bowel cancers have different numbers of chromosomes and we know now that chromosomal instability is present in most solid tumours (90%).

Knowing it happens is one thing: being able to track it in real time to see how it happens is another. This difficulty has recently been overcome by Ana C. F. Bolhaqueiro and her colleagues from the Universities of Utrecht and Groningen who took human colorectal tumour cells and grew them in a cell culture system in the laboratory that permits 3D growth — giving rise to clumps of cells called organoids.

Scheme representing how cells grown as a 3D clump (organoid) can be sampled to follow chromosomal changes. Cells were taken from human colon tumours and from adjacent normal tissue and grown in dishes. The cells were labelled with a fluorescent tag to enable individual chromosomes to been seen by microscopy as the cells divided. At time intervals single cells were selected and sequenced to track changes in DNA. From Johnson and McClelland 2019.

Genetic evolution in real time

As the above scheme shows, the idea of organoids is that their cells grow and divide so that at any time you can select a sample and look at what’s happening to its DNA. Furthermore the DNA can be sequenced to pinpoint precisely the genetic changes that have occurred.

It turned out that cancer cells often make mistakes in apportioning DNA between daughter cells whereas such errors are rare in normal, healthy cells.

It should be said that whilst these errors are common in human colon cancers, a subset of these tumours do not show chromosomal instability but rather have a high frequency of small mutations (called microsatellite instability). Another example of how in cancer there’s usually more than one way of getting to the same end.

Building bridges …

The most common type of gross chromosomal abnormality occurs when chromosomes fuse via their sticky ends to give what are called chromatin bridges (chromatin just means DNA complete with all the proteins normally attached to it). Other errors can give rise to a chromosome that’s become isolated — called a lagging chromosome, it’s a bit like a sheep that has wandered off from the rest of the flock. As the cell finally divides and the daughter cells move apart, DNA bridges undergo random fragmentation.

… but where to …

Little is known about how cells deal with aneuploidy and the extent to which it drives tumour development. This study showed that variation in chromosome number depends on the rate at which chromosomal instability develops and the capacity of a cell to survive in the face of changes in chromosome number. More generally for the future, it shows that the organoid approach offers an intriguing opening for exploring this facet of cancer.

Reference

Bolhaqueiro, A.C.F. et al. (2019). Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nature Genetics 51, 824–834.

Lengauer, C. et al., (1997). Genetic instability in colorectal cancers. Nature 386, 623-627.

Johnson, S.C. and McClelland, S.E. (2019). Watching cancer cells evolve. Nature 570, 166-167.

What’s New in Breast Cancers?

 

One of the best-known things about cancer is that it’s good to catch it early. By that, of course, we don’t mean that you should make an effort to get cancer when you’re young but that, if it does arise it’s a good idea to find out before the initial growth has spread to other places in the body. That’s because surgery and drug treatments are very effective at dealing with ‘primary’ tumours — so much so that over 90% of cancer deaths are caused by cells wandering away from primaries to form secondary growths — a process called metastasis — that are very difficult to treat.

The importance of tumour spreading is shown by the figures for 5-year survival rates. Overall in the USA it’s 90% but this figure falls to below 30% for cancers that have metastasized (e.g., to the lungs, liver or bones). For breast cancer the 5-year survival rate is 99% if it is first detected only in the breast (most cases (62%) are diagnosed at this stage). If it’s spread to blood and lymph vessels in the breast the 5-year survival rate is 85%, dropping to 27% if it’s reached distant parts of the body.

What’s the cause of the problem?

The other thing most people know about cancers is that they’re caused by damage to our genetic material — DNA — that is, by mutations. This raises the obvious notion that secondary tumours might be difficult to deal with because they have accumulated extra mutations compared with those in primaries. And indeed, there have been several studies pointing to just that.

Very recently, however, François Bertucci, Fabrice André and their colleagues in various institutes in France, Switzerland and the USA have mapped in detail the critical alterations in DNA that accumulate as different types of breast cancers develop from early tumours to late, metastatic forms. As is the way these days, their paper contains masses of data but the easiest form of the message comes in the shape of ‘violin plots’. These show the spread of results  — in this case the number of mutations per length of DNA.

Metastatic tumours have a bigger mutational load than early tumours. These plots are for one type of breast tumour (HR+/HER2−) and show results for 381 metastases and 501 early tumours. Red dots = median values: these are the “middle” values rather than an average (or mean) and they show a clear upwards shift in burden as early tumours evolve into metastases. From Bertucci et al., 2019.

The violin plots above are for one subtype of breast cancer (HR+/HER2−). Recall that breast tumours are often defined by which of three types of protein can be detected on the surface of the cells: these are ‘receptors’ that have binding sites for the hormones estrogen and progesterone and for human epidermal growth factor. Hence they are denoted as hormone receptors (HRs) and (human) epidermal growth factor receptor-2 (HER2). Thus tumours may have HRs and HER2 (HR+, HER2+) or various receptors may be undetectable. Triple negative breast cancer (TNBC) is an absence of receptors for both estrogen and progesterone and for HER2.

The plots clearly show an increase in mutation load with progression from early to metastatic tumours (on average from 2.4 to 3.8 mutations per megabase of DNA). Looking at individual genes, nine ‘drivers’ emerged that were more frequently mutated in HR+/HER2− metastatic breast cancers (we described ‘driver’ and ‘passenger’ mutations in Taking Aim at Cancer’s Heart).

So what?

For now these findings give us just a little more insight into what goes on at the molecular level to turn a primary into a metastatic tumour. The fact that some of the acquired driver mutations are associated with poor patient survival offers some guidance as to treatment options.

Don’t get carried away

It’s a familiar story in this field: another small advance in piecing together the jigsaw that is cancer. It doesn’t offer any immediate advance in treatment — mainly because most of the nine ‘driver’ genes identified are tumour suppressors — i.e. they normally act as brakes on cell growth. Mutations knock out that activity and at the moment there is no therapeutic method for reversing such mutations. (The other main class of cancer promoters is ‘oncogenes‘ in which mutations cause hyper-activity).

But such steps are important. The young slave girl in Uncle Tom’s Cabin gave us the phrase “grew like Topsy” — meaning unplanned growth. Cancer growth is indeed unplanned and a bit like Topsy but it’s driven by molecular forces and only through untangling these can we begin to design therapies in a rational way.

Reference

Bertucci, F. et al. (2019). Genomic characterization of metastatic breast cancers. Nature 569, 560–564.

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.

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.