Holiday Reading (4) – Can We Make Resistance Futile?

For those with a fondness for happy endings we should note that, despite the shortcomings of available drugs, the prospects for patients with a range of cancers have increased significantly over the latter part of the twentieth century. The overall 5-year survival rate for white Americans diagnosed between 1996 and 2004 with breast cancer was 91%; for prostate cancer and non-Hodgkin’s lymphoma the figures were 99% and 66%, respectively. These figures are part of a long-term trend of increasingly effective cancer treatment and there is no doubt that the advances in chemotherapy summarised in the earlier Holiday Readings are contributory factor. Nonetheless, the precise contribution of drug treatments remains controversial and impossible to disentangle quantitatively from other significant factors, notably earlier detection and improved surgical and radiotherapeutic methods.

Peering into the future there is no question that the gradual introduction of new anti-cancer drugs will continue and that those coming into use will be more specific and therefore less unpleasant to use. By developing combinations of drugs that can simultaneously poke the blancmange at several points it may be possible to confront tumor cells with a multiple challenge that even their nimbleness can’t evade, thereby eliminating the problem of drug resistance. Perhaps, therefore, in 20 years time we will have a drug cabinet sufficiently well stocked with cocktails that the major cancers can be tackled at key stages in their evolution, as defined by their genetic signature.

However, on the cautionary side we should note that in the limited number of studies thus far the effect of drug combinations on remission times has not been startling, being measured in months rather than years or decades. Having noted the durability of cancer cells we should not be surprised by this and the concern, of course, is that, however ingenious our efforts to develop drug cocktails, we may always come second to the adaptability of nature.

Equally perturbing is the fact that over 90% of cancer deaths arise from primary tumors spreading to other sites around the body. For this phenomenon, called metastasis, there are currently very few treatment options available and the magnitude of this problem is reflected in the fact that for metastatic breast cancer there has been little change in the survival rates over the past forty years.

Metastasis therefore remains one of the key cancer challenges. It’s over 125 years since the London physician Stepen Paget asked the critical question: ‘What is it that allows tumour cells to spread around the body?’ and it’s a daunting fact that only very recently have we made much progress towards an answer – and thus perhaps a way of controlling it. To the fore in this pursuit has been David Lyden and his colleagues at Weill Cornell Medical College in New York. Their most astonishing finding is that cells in the primary tumour release messengers into the circulation and these, in effect, tag what will become landing points for wandering cells. Astonishing because it means that these sites are determined before any tumour cells actually set foot outside the confines of the primary tumour. Lyden has christened this ‘bookmarking’ cancer. That is a quite remarkable finding – but, as ever in science, it merely shifts the question to ‘OK but what’s the messenger?’

A ray of sunshine

It might appear somewhat churlish, especially after all that funding, to end on a note of defeatist gloom so let’s finish with my ray of sunshine that represents a radical approach to the problem. It relies on the fact that small numbers of cells break away from tumors and pass into the circulation. In addition, tumours can release both DNA and small sacs – like little cells – that contain DNA, proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes – a special form of messengers, communicating with other cells by fusing to them. By transferring molecules between cells, exosomes may play a role in mediating the immune response and they are now recognized as key regulators of tumour growth and metastasis.

Step forward Lyden and friends once more who have just shown in a mouse model of pancreatic cancer that exosomes found their way to the liver during the tumour’s earliest stages. Exosomes are taken up by some of the liver cells and this sets off a chain of cell-to-cell signals that eventually cause the accumulation of a kind of molecular glue (fibronectin). This is the critical ingredient in a microenvironment that attracts tumour cells and promotes their growth as a metastasis (secondary growth). So you can think of exosomes as a kind of environmental educator.

Exosome Fig

Exosomes released from primary tumours can mark a niche for metastasis.

The small sacs of goodies called exosomes are carried to the liver where they fuse with some cells, setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells. (from Costa-Silva, B., Lyden, D. et al., Nature Cell Biology 17, 816–826, 2015).

The recent, remarkable technical advances that permit the isolation of exosomes also make it possible to fish out circulating tumour cells and tumour DNA from a mere teaspoonful of blood.

Circulating tumour cells have already been used to monitor patient responses to chemotherapy – when a treatment works the numbers drop: a gradual rise is the earliest indicator of the treatment failing. Even more exciting, this approach offers the possibility of detecting the presence of cancers years, perhaps decades, earlier than can presently be achieved. Coupling this to the capacity to sequence the DNA of the isolated cells to yield a genetic signature of the individual tumor can provide the basis for drug treatment. There are still considerable reservations attached to this approach but if it does drastically shift the stage at which we can detect tumors it may also transpire that their more naïve forms, in which fewer mutations have accumulated, are more susceptible to inhibitory drugs. If that were to be the case then even our currently rather bare, though slowly expanding, drug cabinet may turn out to be quite powerful.

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.

Spray Painting Cancer

I’m pretty certain that anyone reading this will be fully aware that one of the biggest problems in cancer is spotting the blighters. We have, of course, X-ray detection (as in mammography), CTs and MRI scans, all so familiar we need not bother to define them, and there’s also a variety of sampling methods for specific cancers (e.g., the Pap test for cervical cancer). But, useful though all these are, the plain fact of the matter is that none are ideal and in particular the pictures created by imaging methods are very limited in sensitivity. Put another way, they won’t pick something up until it is quite large – a centimeter in diameter – meaning that the abnormal growth is already quite advanced.

Cunning Chemistry

Needless to say, much inspiration and perspiration is being applied to this matter and what has been really exciting over the last ten years or so is the way very smart chemists are collaborating with clinicians to come up with new ways of looking at the problem. One of these clever tactics is being developed in the University of Tokyo using a different type of imaging ‘reporter’ that signals its presence by fluorescing. Fluorescence occurs when a molecule absorbs light and becomes ‘excited’ before relaxing back to its ‘ground state’ by giving off a photon. Fluorescent molecules (fluorophores) are much used in biology because the background signal is often very low so the high signal-to-noise ratio gives excellent sensitivity.

Spray Paint scheme

The cell-surface enzyme GGT converts the small molecule  gGlu-HMRG to a fluorescent form (HMRG) that is then taken up by the cell. GGT is only found on tumor cells so they light up and normal cells do not

Fortunately we don’t need to know how the chemists did it – merely to say that Yasuteru Urano and his colleagues came up with a small molecule (called gGlu-HMRG for short) that does not give off light until a small fragment is chopped off its end, whereupon it changes shape: this flips the switch that turns on fluorescence. The cutting step needs an enzyme that is found on the surface of various cancer cells but not in normal tissue (GGT for short).

Joining Forces

To show that there was real mileage in their idea they followed the time-honored blue-print of cancer research, showing first that it works on tumor cells grown in the lab (and, equally important, that it doesn’t highlight normal cells), before moving to mouse models of ovarian tumors. The later is where chemists meet clinicians because an endoscope is required (quite a small one) – a flexible tube for looking inside the body – devices now so sophisticated that they can incorporate a fluorescence camera.

In the final synthetic step the cunning chemists formulated a spray-on version of their probe molecule so that it can be dispensed during endoscopy or surgery – a bit like an underarm deodorant. Now it’s easy: find suspect tissue, give it a squirt of gGlu-HMRG, wait a few minutes and see if it lights up. The answer is, of course, that in their ovarian cancer model the spray-on graffiti lights up within 10 minutes of sticking to a tumor cell and can detect clumps of cells as small as 1 millimeter in diameter – a terrific advance in terms of sensitivity. The brief time taken for the signal to be visible after the probe has been applied means that within the same procedure it could be used to guide surgeons in removing small tumor masses.

The Tokyo system is not the only one under development. My colleague Andre Neves at the Cambridge Cancer Centre, another of these fiendishly clever chemists, is working on a parallel line using different fluorophores that can be topically applied to the lining of the intestine. The goal here is, of course, the early detection of colon tumors. Yet other approaches use molecules that accumulate preferentially in tumor cells and respond to light in the near-infrared region of the spectrum (800 nm to 2500 nm wavelength, compared to just under 500 nm for gGlu-HMRG), giving an even better signal-to-noise ratio.

This is, as Mr. Churchill might have pointed out, not even the beginning of the end of this story. But it is one more small and innovative step forward. Not all cancers even of the same type will be detectable by a given probe because they vary so much in the genes they express but the ingenuity of the chemists gives hope that a substantial panel of ever more sensitive reporters will emerge. It is also true that endoscopy is unlikely to gain widespread popularity as a routine screening method. However, these advances, moving us to detection at ever earlier stages may become very powerful as a follow-up test, combined with the capacity for simultaneous treatment, when tumor cells have been detected in more comfortable screens, for example as circulating cells in small blood samples, an immensely exciting prospect to which we will return in a later episode.

 References

Urano, Y., Masayo Sakabe, Nobuyuki Kosaka, Mikako Ogawa, Makoto Mitsunaga, Daisuke Asanuma, Mako Kamiya, Matthew R. Young, Tetsuo Nagano, Peter L. Choyke, and Kobayashi, H. (2011). Rapid Cancer Detection by Topically Spraying a γ-Glutamyltranspeptidase–Activated Fluorescent Probe. Science Translational Medicine 3, 110ra119.

http://www.ncbi.nlm.nih.gov/pubmed/22116934

Shi, C. (2012). Comment on “Rapid Cancer Detection by Topically Spraying a γ-Glutamyltranspeptidase–Activated Fluorescent Probe. Science Translational Medicine 4, 121le1.

http://stm.sciencemag.org/content/4/121/121le1.long