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.”


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


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.”


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.


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.


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.