One More Small Step

 

Back in the nineteenth century a chap called Augustus De Morgan came up with a set of laws that, when explained in English, sound like the lyrics of a Flanders & Swann song. Opaque to non-maths nerds they may be but they helped to build the mathematics of logic, so next time you meet AND / OR gates in electronics, spare him a thought.

In fact Augustus is rare — maybe unique — among mathematicians in that he’s not completely forgotten, for it was he who penned the lines:

Big fleas have little fleas upon their backs to bite ’em,
And little fleas have lesser fleas, and so, 
ad infinitum.

Given that we now know there’s over 2,500 species of fleas ranging in size from tiny to nearly one centimeter long, it may be literally true. But here, for once, the truth doesn’t matter. It’s a silly rhyme but nonsense verse it is not for it could well serve as a motto for biology because it really captures the essential truth of life: the exquisite choreography of living systems by which incomprehensible numbers of interactions come together to make them work.

Human fleas. Don’t worry: you’ll know if you have them.

Unbidden, De Morgan’s ditty came into my head as I was reading the latest research paper from David Lyden’s group, which he very kindly sent me ahead of publication this week. Avid readers will know the name for we have devoted several episodes (Keeping Cancer Catatonic, Scattering the Bad Seed and Holiday Reading (4) – Can We Make Resistance Futile) to the discoveries of his group in tackling one of the key questions in cancer — namely, how do tumour cells find their targets when they spread around the body? Key because it is this process of ‘metastasis’ that causes most (over 90%) of cancer deaths and if we knew how it worked maybe we could block it.

A succinct summary of those already condensed episodes would be: (1) cells in primary tumours release ‘messengers’ into the circulation that ‘tag’ metastatic sites before any cells actually leave the tumour, (2) the messengers that do the site-tagging are small sacs — mini cells — called exosomes, and (3) they find specific addresses by carrying protein labels (integrins) that home in to different organs — we represented that in the form of a tube train map in Lethal ZIP codes that pulled the whole story together.

The next small step

Now what the folks from Weill Cornell Medicine, New York, Sloan Kettering and a host of other places have done is adapt a flow system to look more closely at exosomes.

Separating small bodies. Particles are injected into a flowing liquid (left) and cross flow at right angles through a membrane (bottom) permits separation on the basis of effective size (called asymmetrical flow field-flow fractionation).

They found that a wide variety of tumour cell types secrete two distinct populations of exosomes — small (60-80 nanometres diameter) and large (90-120 nm). What’s more they found a third type of nanoparticle, smaller than exosomes (less than 50 nm) and without a membrane — so it’s a kind of blob of lipids and proteins (a micelle would be a more scientific term) — that they christened exomeres.

Is it real?

A perpetual problem in biology is reproducibility — that is, whether a new finding can be replicated independently by someone else. Or, put more crudely, do I believe this? This is such an important matter that it’s worth a separate blog but for the moment we’re OK because the results in this paper speak for themselves. First, by using electron microscopy, Lyden et al could actually look at what they’d isolated and indeed discerned three distinct nano-populations — which is how they were able to put the size limits on them.

Electron microscopy of (left) the input mixture (pre-fractionation) and separated fractions: exomere, small exosomes and large exosomes released by tumour cells.. Arrows indicate exomeres (red), small exosomes (blue) and large exosomes (green), from Zhang et al. 2018.

But what’s most exciting in terms of the potential of these results is what’s in the packets. Looking at the fats (lipids), proteins and nucleic acids (DNA and RNA) they contained it’s clear that these are three distinct entities — which makes it very likely they have different effects.

Given their previous finding it must have been a great relief when Lyden & Co identified integrin address proteins in the two exosome sub-populations. But what’s really astonishing is the range of proteins born by these little chaps: something like 400 in exomeres, about 1000 in small exosomes and a similar number in the big ones — and the fact that each contained unique sets of proteins. The new guys — exomeres — carry among other proteins, metabolic enzymes so it’s possible that when they deliver their cargo it might be able to change the metabolic profile of its target. That could be important as we know such changes happen in cancer.

It’s a bewildering picture and working out even the basics of what these little guys do and how it influences cancer is, as we say, challenging. But I think I know a good man for the job!

Augustus De Morgan looking down.

Mathematicians have a bit of a tendency to look down on us experimentalists thrashing around in the undergrowth and I suspect that up in the celestial library, as old Augustus De Morgan thumbed through this latest paper, a slight smile might have come over his face and he could have been heard to murmur: “See, I told you.”

References

Zhang, H. et al. (2018). Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nature Cell Biology 20, 332–343. doi:10.1038/s41556-018-0040-4

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Lethal ZIP codes

In Keeping Cancer Catatonic we retailed how, over 125 years ago, the London physician Stephen Paget came up with his ‘seed and soil’ idea to explain why it was that when cancers spread to distant sites around the body by getting into the circulation they didn’t simply stick to the first tissue they came across. Paget had spotted that cancers tend to have preferred sites for spreading: tumours of the eye tend to travel to the liver, rather than the much handier brain, and breast cancers, Paget’s speciality, commonly spread to the liver but also to the lungs, kidneys, spleen and bone. So his idea was that certain distant secondary sites are somehow made more receptive to tumor growth, just as soil can be prepared for seeds to sprout.

So the key question became ‘how?’ and it’s hung in the cancer air for well over a century during which we’ve made very little progress towards an answer – and it is crucial because the business of tumour cells spreading (metastasizing) causes most cancer deaths (over 90%).

But, at long last, things have started to move, largely due to the efforts of David Lyden and his colleagues at Weill Cornell Medical College. Their first astonishing contribution was to show that cells in primary tumours release messengers into the circulation and these, in effect, tag what will become landing points for wandering tumour cells – i.e., the target sites are determined before any tumour cells actually set foot outside the confines of the primary tumour.

After that seismic revelation the story advanced a step further (in Scattering the Bad Seed) with some molecular detail of how the sites are marked – an effect Lyden has christened ‘Bookmarking cancer’ – and how when tumour cells do settle in their new niche they may be kept dormant for many years before starting to expand.

Carrying the flag

The next chapter in the story, as retailed in Holiday Reading (4) – Can We Make Resistance Futile?, revealed that the message is carried by small sacs – like little cells – called exosomes that are released from tumour cells. These float around the circulation until they find their target site, whereupon they plant the flag by setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells.

That is all truly amazing stuff but, as we noted in Holiday Reading (4) – Can We Make Resistance Futile?, a recurring theme in science is that one answer merely poses the next question – in this case ‘what’s the messenger?’

As in all the best thrillers, the authors have kept us in suspense to the last, helped presumably by their not knowing the answer. But in this week’s Nature (Oct. 28, 2015) comes the denoument to this whodunit.

Mister postman look and see …

Many moons ago an outfit called the Marvelettes had a No. 1 hit with Please Mr. Postman and somewhat later the Fab Four did a re-hash that met with equal success. Perhaps we should have asked them how nature would go about directing little packages around the body. John, Ringo and the lads would, with their earthy, Liverpudlian logic, have pointed out the triviality of the problem of exosome addressing. ‘It’s not like you’re sending stuff all over the world, is it? You’ve only got a few targets – the major organs of the body. So a dead simple code will do. You know your messengers are proteins – ’coz they do everything – OK? So, pick a protein that comes in two bits with a few variants of each: mix and match and there’s yer postcodes. Now … what was that ditty about yellow subsurface vessels …’

And so it came to pass …

And the messenger is …

A family of proteins called integrins whose job is to span the membranes of cells, thereby promoting cell-cell interactions. They are indeed made of two different chains stuck together (called α (alpha) and β (beta)) and the upshot is that our cells can make about 24 unique integrins – more than enough to form a coded address system to direct tumour cells around the body. Well done lads!

What Ayuko Hoshino, David Lyden and their many collaborators did was to tag exosomes released from various types of cancer cell with a fluorescent dye and inject them into mice. The fluorescent label enabled them to track the exosomes and it turned out that, for a variety of cancer cells (breast, pancreatic, colorectal, lung, melanoma and pediatric) the exosomes travelled to the organs associated with metastasis (e.g., breast cancer exosomes stuck in the lungs, pancreatic cancer exosomes in the liver, etc). In other words exosome spread mimicked the pattern of the tumour from which they were derived. Once they had landed the exosomes set about reprogramming the organ sites to make a fertile microenvironment capable of supporting tumor cell growth in a new colony.

When they looked at the exosome proteins they found a particular member of the integrin family flagged each organ-specific site. Thus α6β4 promotes lung metastasis, αvβ5 homes in on the liver, αvβ3 on the brain, etc.

MapFinding a home

To spread around the body (metastasise) primary tumours first release small sacs (exosomes) carrying protein tags (integrins). Moving through the circulatory system the integrin tags home in to specific addresses found on different organs. The effect of exosomes sticking to target sites is to prepare the ground for cells released by the tumour to adhere and colonise.

Down the tube

You could think of primary tumours as being a bit like us when we move to a new city and try to find a des. res. in a place you don’t know. We could just ramble round the subway system until something catches our eye but that might take for ever. Much more efficient is to ask someone with local knowledge where would be good spots to target. For disseminating tumours their exosomes are the scouts who do the foot-slogging: the protein signatures on the surface of these small, tumour-secreted packages home in on postcodes that define a desirable locale for metastatic spread.

Shooting the messenger

An obvious question is ‘If exosomes are critical in defining metastatic sites, can you block their action – and what happens when you do?’ In preliminary experiments Hoshino & Co showed that either knockdown of specific integrins or blocking the capacity of these proteins to stick to their targets (with a specific antibody or short synthetic peptides) significantly reduced exosome adhesion, thereby blocking pre-metastatic niche formation and liver metastasis.

A new beginning?

We described these fabulous results as the denouement but, of course, it isn’t. As Mr. Churchill remarked in a somewhat different context: ‘Now this is not the end.’ It is rather a step to answering an old question but it’s incredibly exciting. If screening for exosomes leads to the detection of cancer not just years but perhaps decades earlier than can be achieved by present methods and if blocking their action can keep metastasis at bay, then the field of cancer will be utterly transformed.

References

Hoshino, A. et al. (2015). Tumour exosome integrins determine organotropic metastasis. Nature doi:10.1038/nature15756.

Ruoslahti, E. (1996). RGD and Other Recognition Sequences for Integrins. Annual Review of Cell and Developmental Biology 12, 697-715.

Seeing the Invisible: A Cancer Early Warning System?

Sherlock Holmes enthusiasts who also follow this column may, in a contemplative moment, have asked themselves whether their hero would have made a good cancer detective. Answer perhaps ‘yes’ in that he was obsessive about sticking to the facts and not guessing and would probably have said that, when tracking down a secretive quarry, you need to be as open-minded as possible in looking for clues. One of his most celebrated efforts at marrying observation with knowledge was his greeting upon first meeting Dr. Watson: “How are you? You have been in Afghanistan, I perceive”. Watson was suitably astonished by this apparent clairvoyance although its basis was in fact rather mundane and only beyond him because, as Sherlock kindly explained, “You see, but you do not observe.”

Holmes-Image-Loupe

Dr. Holmes perchance?

If Watson had paused to wonder whether Holmes’ combination of superiority complex and investigative genius would have fitted him for a career in the medical fraternity, he might have reflected that indeed many internal afflictions do manifest external signs – much as the furtive body language of a felon on a job might mark him out to the observant eye in the throng of bodies pressing into Baker Street underground station. So perhaps the ’tec turned doc could make it in infectious diseases or become a consultant in rheumatoid arthritis. But would he have steered clear of oncology, reasoning that most cancers are without symptoms during their early development and that even he could not observe the invisible?

Lithograph of Baker Street Station   Baker Street Station on the Metropolitan Railway in 1863 (London Transport Museum collection)

Probably, but before taking that decision he would have asked for a tutorial – perhaps from that bright fellow Stephen Paget, who would have explained that cancers are unusual lumps of cells that can often be cut out by surgeons such as himself. But he’d have highlighted the problem that similar growths commonly turn up later at other, secondary, sites in the body – they are what kills most cancer patients and no one has a clue how this happens or what to do about it. Holmes would doubtless have taken a deep suck on his pipe, commented that, as no one appeared to disagree with William Harvey’s 250 year old finding that blood is passed to every nook and cranny of the body by the circulatory system, it scarcely required his giant intellect to deduce that to be the most probable way of spreading tumours. Further observing that cancers develop very slowly, he would have pointed out that it is highly likely that within the body there might be clues – molecular signs that something is amiss – long before overt disease appears. All that was required was a biological magnifying glass and tweezers to spot and pick out rogue cells and molecules. Muttering ‘Elementary’ he would then have asked to be excused to return to the really tricky problem of outsmarting Professor Moriarty.

An Achilles’ heel?

Well, as we have just reviewed in Scattering the Bad Seed, some 130 years after that imaginary encounter the ‘elementary’ way in which tumours spread to form metastases is just beginning to be revealed and, of course, the hope is that eventually this knowledge will lead to ways of treating disseminated cancers or even preventing them. That’s a wonderful prospect but even more exciting are technical advances enabling us to exploit what Sherlock had spotted as something of a cancer Achilles’ heel – namely that, if tumour cells spread via the bloodstream, we need only the right tools (magnifying glass and tweezers) to detect secondary growths almost before they’ve started to form. As most people know, the earlier cancers are caught the more likely they are to be cured, the most critical intervention being before they have spread to form metastases that are the major cause of death.

The things you find in blood

In fact, quite apart from intact tumour cells migrating around the circulation, it’s been known for 40 years that most types of cell in our bodies have the rather odd quirk of releasing short bits of their DNA into the circulation. Cancer cells do this too and these chromosome fragments reflect the genetic mayhem that is their hallmark. How DNA gets out of the nucleus and then across the outer membrane of the cell isn’t known but it does – and the bits of nucleic acid act as messengers, being taken up by other cells that respond by changing their behaviour. In Beware of Greeks we saw that DNA fragments released by leukemia cells can help those cells escape from the bone marrow into circulating blood.

There’s yet another sort of cellular garbage swishing around in our circulation: small sacs like little cells that contain proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes and in fact they’re not at all rubbish but are also messengers, communicating with other cells by fusing and transferring their contents. So exosomes are another form of environmental educator.

Going fishing

The problem has been that until very recently it has not been possible to fish out tumour cells or DNA from the vast number of cells in blood (we’ve each got over 20 trillion red blood cells in our five litres or so). However, an exciting new development has been the application of silicon chip technology to the detection of circulating tumour cells (CTCs). The chips, which are the size of a microscope slide (10 x 2 cm), have about 80,000 microscopic columns etched on their surface that are coated with an array of antibodies that stick to molecules expressed on the surface of CTCs. By incorporating the chips into small flow cells it’s possible to capture about 100 CTCs from a teaspoon of blood – that’s pulling out one tumour cell from a background of a billion (109) normal cells.

CTC CHIP

Tumour cell isolation from whole blood by a CTC-chip. Whole blood is circulated through a flow cell containing the capture columns (Stott et al., 2010)

This microfluidics approach can also be used to isolate tumour cell DNA. For this the coatings are short stretches of artificial DNA of different sequences: these bind to free DNA in the same way that two strands of DNA stick together to make the double helix.

This remarkable technology may offer both the most promising way to early tumour detection and of determining responses to drugs. It also provides a bridge between proteomic and genomic technologies because DNA, captured directly or extracted from isolated cells, can be used for whole genome sequencing. If this system is able to capture cells from most major types of tumour it will indeed provide a rapid route from early detection through genomic analysis to tailored chemotherapy without the requirement for tumour biopsies. In Signs of Resistance we noted that it’s possible to track the response of secondary tumours (metastases) to drug treatment (chemotherapy) using this method of pulling out tumor DNA from blood and sequencing it.

The really optimistic view is that chip isolation of DNA or tumour cells may be a means to cancer detection years, perhaps decades, before any other test would show its presence. By following up with the power of sequencing, the hope is that appropriate drug cocktails can be devised to, so to speak, nip the tumour in the bud.

Wizard’s secret

By the way, Conan Doyle eventually revealed the method behind Sherlock’s wizardry: Watson was a medical man but walked with a military bearing: the skin on his wrists was fair but his face tanned and haggard and he held his left arm in a stiff and unnatural manner. So here was a British army doctor who had served in the tropics (or somewhere equally hot) and been wounded. In 1886 where would that have been? Oh yes, of course. Afghanistan.

Reference

Stott, S.L., Hsu, C.-H., Tsukrov, D.I., Yu, M., Miyamoto, D.T., Waltman, B.A., Rothenberg, M.S., Shah, A.M., Smas, M.E., Korir, G.K., Floyd, Jr., F.P., Gilman, A.J., Lord, J.B., Winokur, D., Springer, S., Irimia, D., Nagrath, S., Sequist, L.V., Lee, R.J., Isselbacher, K.J., Maheswaran, S., Haber, D.A. and Toner, M. (2010). Isolation of circulating tumour cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences of the United States of America 107, 18392-18397.