I Know What I Like

 

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

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

Can science help?

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

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

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

Step this way: entrance free

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

  1. Cells grown in culture:

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

 

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

 

 

 

 

 

 

3. Waves of calcium in firing neurons:

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

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

 

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

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

 

5. The restless cell (1):

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

 

6. The restless cell (2):

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

 

7. Cell division:

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

 

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

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

 

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

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

It’s certainly science – but is it art?

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

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

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

Controversial?

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

Lorenzo’s Oil for Nervous Breakdowns

 

A Happy New Year to all our readers – and indeed to anyone who isn’t a member of that merry band!

What better way to start than with a salute to the miracles of modern science by talking about how the lives of a group of young boys have been saved by one such miracle.

However, as is almost always the way in science, this miraculous moment is merely the latest step in a long journey. In retracing those steps we first meet a wonderful Belgian – so, when ‘name a famous Belgian’ comes up in your next pub quiz, you can triumphantly produce him as a variant on dear old Eddy Merckx (of bicycle fame) and César Franck (albeit born before Belgium was invented). As it happened, our star was born in Thames Ditton (in 1917: his parents were among the one quarter of a million Belgians who fled to Britain at the beginning of the First World War) but he grew up in Antwerp and the start of World War II found him on the point of becoming qualified as a doctor at the Catholic University of Leuven. Nonetheless, he joined the Belgian Army, was captured by the Germans, escaped, helped by his language skills, and completed his medical degree.

Not entirely down to luck

This set him off on a long scientific career in which he worked in major institutes in both Europe and America. He began by studying insulin (he was the first to suggest that insulin lowered blood sugar levels by prompting the liver to take up glucose), which led him to the wider problems of how cells are organized to carry out the myriad tasks of molecular breaking and making that keep us alive.

The notion of the cell as a kind of sac with an outer membrane that protects the inside from the world dates from Robert Hooke’s efforts with a microscope in the 1660s. By the end of the nineteenth century it had become clear that there were cells-within-cells: sub-compartments, also enclosed by membranes, where special events took place. Notably these included the nucleus (containing DNA of course) and mitochondria (sites of cellular respiration where the final stages of nutrient breakdown occurs and the energy released is transformed into adenosine triphosphate (ATP) with the consumption of oxygen).

In the light of that history it might seem a bit surprising that two more sub-compartments (‘organelles’) remained hidden until the 1950s. However, if you’re thinking that such a delay could only be down to boffins taking massive coffee breaks and long vacations, you’ve never tried purifying cell components and getting them to work in test-tubes. It’s a process called ‘cell fractionation’ and, even with today’s methods, it’s a nightmare (sub-text: if you have to do it, give it to a Ph.D. student!).

By this point our famous Belgian had gathered a research group around him and they were trying to dissect how insulin worked in liver cells. To this end they (the Ph.D. students?!) were using cell fractionation and measuring the activity of an enzyme called acid phosphatase. Finding a very low level of activity one Friday afternoon, they stuck the samples in the fridge and went home. A few days later some dedicated soul pulled them out and re-measured the activity discovering, doubtless to their amazement, that it was now much higher!

In science you get odd results all the time – the thing is: can you repeat them? In this case they found the effect to be absolutely reproducible. Leave the samples a few days and you get more activity. Explanation: most of the enzyme they were measuring was contained within a membrane-like barrier that prevented the substrate (the chemical that the enzyme reacts with) getting to the enzyme. Over a few days the enzyme leaked through the barrier and, lo and behold, now when you measured activity there was more of it!

Thus was discovered the ‘lysosome’ – a cell-within-a cell that we now know is home to an array of some 40-odd enzymes that break down a range of biomolecules (proteinsnucleic acidssugars and lipids). Our self-effacing hero said it was down to ‘chance’ but in science, as in other fields of life, you make your own luck – often, as in this case, by spotting something abnormal, nailing it down and then coming up with an explanation.

In the last few years lysosomes have emerged as a major player in cancer because they help cells to escape death pathways. Furthermore, they can take up anti-cancer drugs, thereby reducing potency. For these reasons they are the focus of great interest as a therapeutic target.

Lysosomes in cells revealed by immunofluorescence.

Antibody molecules that stick to specific proteins are tagged with fluorescent labels. In these two cells protein filaments of F-actin that outline cell shape are labelled red. The green dots are lysosomes (picked out by an antibody that sticks to a lysosome protein, RAB9). Nuclei are blue (image: ThermoFisher Scientific).

Play it again Prof!

In something of a re-run of the lysosome story, the research team then found itself struggling with several other enzymes that also seemed to be shielded from the bulk of the cell – but the organelle these lived in wasn’t a lysosome – nor were they in mitochondria or anything else then known. Some 10 years after the lysosome the answer emerged as the ‘peroxisome’ – so called because some of their enzymes produce hydrogen peroxide. They’re also known as ‘microbodies’ – little sacs, present in virtually all cells, containing enzymatic goodies that break down molecules into smaller units. In short, they’re a variation on the lysosome theme and among their targets for catabolism are very long-chain fatty acids (for mitochondriacs the reaction is β-oxidation but by a different pathway to that in mitochondria).

Peroxisomes revealed by immunofluorescence.

As in the lysosome image, F-actin is red. The green spots here are from an antibody that binds to a peroxisome protein (PMP70). Nuclei are blue (image: Novus Biologicals)

Cell biology fans will by now have worked out that our first hero in this saga of heroes is Christian de Duve who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and George Palade.

A wonderful Belgian. Christian de Duve: physician and Nobel laureate.

Hooray!

Fascinating and important stuff – but nonetheless background to our main story which, as they used to say in The Goon Show, really starts here. It’s so exciting that, in 1992, they made a film about it! Who’d have believed it?! A movie about a fatty acid!! Cinema buffs may recall that in Lorenzo’s Oil Susan Sarandon and Nick Nolte played the parents of a little boy who’d been born with a desperate disease called adrenoleukodystrophy (ALD). There are several forms of ALD but in the childhood disease there is progression to a vegetative state and death occurs within 10 years. The severity of ALD arises from the destruction of myelin, the protective sheath that surrounds nerve fibres and is essential for transmission of messages between brain cells and the rest of the body. It occurs in about 1 in 20,000 people.

Electrical impulses (called action potentials) are transmitted along nerve and muscle fibres. Action potentials travel much faster (about 200 times) in myelinated nerve cells (right) than in (left) unmyelinated neurons (because of Saltatory conduction). Neurons (or nerve cells) transmit information using electrical and chemical signals.

The film traces the extraordinary effort and devotion of Lorenzo’s parents in seeking some form of treatment for their little boy and how, eventually, they lighted on a fatty acid found in lots of green plants – particularly in the oils from rapeseed and olives. It’s one of the dreaded omega mono-unsaturated fatty acids (if you’re interested, it can be denoted as 22:1ω9, meaning a chain of 22 carbon atoms with one double bond 9 carbons from the end – so it’s ‘unsaturated’). In a dietary combination with oleic acid  (another unsaturated fatty acid: 18:1ω9) it normalizes the accumulation of very long chain fatty acids in the brain and slows the progression of ALD. It did not reverse the neurological damage that had already been done to Lorenzo’s brain but, even so, he lived to the age of 30, some 22 years longer than predicted when he was diagnosed.

What’s going on?

It’s pretty obvious from the story of Lorenzo’s Oil that ALD is a genetic disease and you will have guessed that we wouldn’t have summarized the wonderful career of Christian de Duve had it not turned out that the fault lies in peroxisomes.

The culprit is a gene (called ABCD1) on the X chromosome (so ALD is an X-linked genetic disease). ABCD1 encodes part of the protein channel that carries very long chain fatty acids into peroxisomes. Mutations in ABCD1 (over 500 have been found) cause defective import of fatty acids, resulting in the accumulation of very long chain fatty acids in various tissues. This can lead to irreversible brain damage. In children the myelin sheath of neurons is damaged, causing neurological defects including impaired vision and speech disorders.

And the miracle?

It’s gene therapy of course and, helpfully, we’ve already seen it in action. Self Help – Part 2 described how novel genes can be inserted into the DNA of cells taken from a blood sample. The genetically modified cells (T lymphocytes) are grown in the laboratory and then infused into the patient – in that example the engineered cells carried an artificial T cell receptor that enabled them to target a leukemia.

In Gosh! Wonderful GOSH we saw how the folk at Great Ormond Street Hospital adapted that approach to treat a leukemia in a little girl.

Now David Williams, Florian Eichler, and colleagues from Harvard and many other centres around the world, including GOSH, have adapted these methods to tackle ALD. Again, from a blood sample they selected one type of cell (stem cells that give rise to all blood cell types) and then used genetic engineering to insert a complete, normal copy of the DNA that encodes ABCD1. These cells were then infused into patients. As in the earlier studies, they used a virus (or rather part of a viral genome) to get the new genetic material into cells. They choose a lentivirus for the job – these are a family of retroviruses (i.e. they have RNA genomes) that includes HIV. Specifically they used a commercial vector called Lenti-D. During the life cycle of RNA viruses their genomes are converted to DNA that becomes a permanent part of the host DNA. What’s more, lentiviruses can infect both non-dividing and actively dividing cells, so they’re ideal for the job.

In the first phase of this ongoing, multi-centre trial a total of 17 boys with ALD received Lenti-D gene therapy. After about 30 months, in results reported in October 2017, 15 of the 17 patients were alive and free of major functional disability, with minimal clinical symptoms. Two of the boys with advanced symptoms had died. The achievement of such high remission rates is a real triumph, albeit in a study that will continue for many years.

In tracing this extraordinary galaxy, one further hero merits special mention for he played a critical role in the story. In 1999 Jesse Gelsinger, a teenager, became the first person to receive viral gene therapy. This was for a metabolic defect and modified adenovirus was used as the gene carrier. Despite this method having been extensively tested in a range of animals (and the fact that most humans, without knowing it, are infected with some form of adenovirus), Gelsinger died after his body mounted a massive immune response to the viral vector that caused multiple organ failure and brain death.

This was, of course, a huge set-back for gene therapy. Despite this, the field has advanced significantly in the new century, both in methods of gene delivery (including over 400 adenovirus-based gene therapy trials) and in understanding how to deal with unexpected immune reactions. Even so, to this day the Jesse Gelsinger disaster weighs heavily with those involved in gene therapy for it reminds us all that the field is still in its infancy and that each new step is a venture into the unknown requiring skill, perseverance and bravery from all involved – scientists, doctors and patients. But what better encouragement could there be than the ALD story of young lives restored.

It’s taken us a while to piece together the main threads of this wonderful tale but it’s emerged as a brilliant example of how science proceeds: in tiny steps, usually with no sense of direction. And yet, despite setbacks, over much time, fragments of knowledge come together to find a place in the grand jigsaw of life.

In setting out to probe the recesses of metabolism, Christian de Duve cannot have had any inkling that he would build a foundation on which twenty-first century technology could devise a means of saving youngsters from a truly terrible fate but, my goodness, what a legacy!!!

References

Eichler, F. et al. (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. The New England Journal of Medicine 377, 1630-1638.

 

Re-writing the Manual of Life

A little while ago we talked about a fantastic triumph by a team at Great Ormond Street Hospital (Gosh! Wonderful GOSH) in using a form of immunotherapy to save a little girl. What they did was to take the T cells from a sample of her blood and use gene editing – molecular cutting and pasting – to remove some genes and add others before growing more of the cells and then putting them back into the patient.

Gene editing – genetic engineering that removes or inserts sections of DNA – uses engineered nucleases, enzymes that snip DNA but do so in a controlled way by homing in on a specific site (i.e. a defined sequence of As, Cs, Gs and Ts).

We mentioned that there are four main ways of doing this kind of engineering – the GOSH group used ‘transcription activator-like effectors’ (TALEs). However, the method that has made the biggest headlines is called CRISPR/Cas, and it has been very much in the news because a legal battle is underway to determine who did what in its development and who, therefore, will be first in line for a Nobel Prize.

Fortunately we can ignore such base pursuits and look instead at where this technology might be taking us.

What is CRISPR/Cas?

CRISPRs (pronounced crispers) are bits of DNA that contain short repetitions of base sequence, each next to a ‘spacer’ sequence. The spacers have accumulated in bacteria as a defence mechanism – they’re part of the bacterial immune system – and they’re identical to sequences found in viruses that infect microbes. In other words, the cunning bugs pick up bits of dangerous viruses to make a rogues gallery so they can recognize and attack those viruses next time they pop in.

Close to CRISPR sit genes encoding Cas proteins (enzymes that cut DNA, so they’re ‘nucleases’). When the CRISPR-spacer DNA is read by the machinery of the cell to make RNA, the spacer regions stick to Cas proteins and the whole complex, including the viral sequences, can roam the cell seeking a virus with genetic material that matches the CRISPR RNA. The CRISPR RNA sticks to the virus and Cas chops its DNA – end of virus. So Cas, by binding to CRISPR RNA, becomes an RNA-guided DNA cutter.

crispr-pic

CRISPR-CAS: Bug defence against invaders. Viruses can attack bacteria just as they can human cells. Over time bugs have evolved a cunning defence strategy: they insert short bits of viral DNA into their own genome (above). These contain repeated sequences of bases and each is followed by short segments of ‘spacer DNA’ (above). This happens next to DNA that encodes Cas proteins so that both are ‘read’ to make RNA (transcription). Cas proteins bind to spacer RNA, leaving the adjacent viral RNA free to attach to any complementary viral DNA it encounters. The Cas enzyme is thus guided to DNA that it can cleave. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

Why is CRISPR/Cas in the headlines?

We saw in Gosh! Wonderful GOSH how the Great Ormond Street Hospital team tinkered with DNA and in Self Help – Part 2 we summarized another way of doing this using viruses (notably a disabled form of the human immunodeficiency virus) to carry novel genes into cells.

A further arm of immunotherapy attempts to reverse an effect called checkpoint blockade whereby the immune system response to tumours is damped down – e.g. by using antibodies that target a protein called PD-1 (Self Help – Part 1).

Now comes news of a Chinese trial which will be the first time cells modified using CRISPR–Cas9 gene editing have been injected into people. The chap in charge is Lu You from Sichuan University’s West China Hospital in Chengdu and the plan is to take T cells from the blood patients with metastatic non-small cell lung cancer for whom chemotherapy, radiation therapy and other treatments have failed.

The target will be the PD-1 gene, the idea being that, if you want to stop PD-1 doing its stuff, far better than mucking about with antibodies is to just knock out its gene: no gene no protein! What could possibly go wrong?

Well, wonderful though CRISPR is, it doesn’t always hit the right target but in this trial the cells can be tested to make sure it’s the PD-1 gene that’s been zonked – so that shouldn’t be a problem. However, it’s a blockbuster in that all the multiplied T cells put back into the patient will be active – i.e. will have lost the PD-1 brake. Whilst that may be good for zonking tumours, goodness knows what it might do elsewhere.

The initial trial is on a small scale – just 10 people. If there are problems one possibility is to try to take the T cells from the site of the tumour, which might select those specifically targeting the tumour – not straightforward as lung cancers are difficult to get at.

Anyone for a DNA upgrade?

It’s hard to say where all this is leading. However, as Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys, the only safe bet is that China will be to the fore.

 

The Shape of Things to Come?

One of the problems of trying to keep up with cancer – and indeed helping others to do so – is that you (i.e. ‘I’) get really irritated with the gentlemen and ladies of the press for going over the top in their efforts to cover science. I have therefore been forced to have a few rants about this in the past – actually, when I came to take stock, even I was a bit shocked at how many. Heading the field were Not Another Great Cancer Breakthough, Put A Cap On It and Gentlemen… For Goodness Sake. And not all of these were provoked by The Daily Telegraph!

If any of the responsible reporters read this blog they probably write me off as auditioning for the Grumpy Old Men tv series. But at least one authoritative voice says I’m really very sane and balanced (OK, it’s mine). Evidence? The other day I spotted the dreaded G word (groundbreaking) closely juxtaposed to poor old Achilles’ heel – and yes, it was in the Telegraph – but, when I got round to reading the paper, I had to admit that the work referred to was pretty stunning. Although, let’s be clear, such verbiage should still be banned.

A Tumour Tour de Force

The paper concerned was published in the leading journal Science by Nicholas McGranahan, Charles Swanton and colleagues from University College London and Cancer Research UK. It described a remarkable concentration of current molecular fire-power to dissect the fine detail of what’s going on in solid tumours. They focused on lung cancers and the key steps used to paint the picture were as follows:

1. DNA sequencing to identify mutations that produced new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ – meaning molecular flags on the cell surface that can provoke an immune response – i.e. the host makes antibody proteins that react with (stick to) the antigens). Typically they found just over 300 of these ‘neoantigens’ per tumour – a reflection of the genetic mayhem that occurs in cancer.

2 tumoursVariation in neoantigen profile between two multi-region sequenced non-small cell lung tumours. There were approximately 400 (left) and 300 (right) neoantigens/tumour

  • Blue: proportion of clonal neoantigens found in every tumour region.
  • Yellow: subclonal neoantigens shared in multiple but not all tumour regions.
  • Red: subclonal (‘private’) neoantigens found in only one tumour region.
  • The left hand tumour (mostly blue, thus highly clonal) responded well to immunotherapy (from McGranahan et al. 2016).

2. Screening the set of genes that regulate the immune system – that is, make proteins that detect which cells belong to our body and which are ‘foreign.’ This is the human leukocyte antigen (HLA) system that is used to match donors for transplants – called HLA typing.

3. Isolating specialised immune cells (T lymphocytes) from samples of two patients with lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

4. Detecting proteins released by different types of infiltrating T cells that regulate the immune response. These include so-called immune checkpoint molecules that limit the extent of the immune response. This showed that T cell subsets that were very good at recognizing neo-antigens – and thus killing cancer cells (they’re CD8+ T cells or ‘killer’ T cells) also made high levels of proteins that restrain the immune response (e.g., PD-1).

5. Showing that immunotherapy (using the antibody pembrolizumab that reacts with PD-1) could significantly extend survival of patients with advanced non-small cell lung cancer. We’ve already met this approach in Self-help Part 1.

The critical finding was that the complexity of the tumour (called the clonal architecture) determines the outcome. Durable benefit from this immunotherapy requires a high level of mutation but a restricted range of neo-antigens. Put another way, tumours that are highly clonal respond best because they have common molecular flags present on every tumour cell.

6. Using the same methods on some skin cancers (melanomas) with similar results.

What did this astonishing assembly of results tell us?

It’s the most detailed picture yet of what’s going on in individual cancers. As one of the authors, Charles Swanton, remarked “This is exciting. This opens up a way to look at individual patients’ tumours and profile all the antigen variations to figure out the best ways for treatments to work. This takes personalised medicine to its absolute limit where each patient would have a unique, bespoke treatment.”

He might have added that it’s going to take a bit of time and a lot of money. But as a demonstration of 21st century medical science it’s an absolute cracker!

References

McGranahan et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).

 

Self Help – Part 1

It’s not easy to find good things to say about cancer and humour is equally elusive, as those of us who lecture on the subject know very well. But most people are aware of one cheering fact: cancers aren’t transmissible between humans – that is, they’re not like ’flu, venereal diseases and lots of other nasty things we pass around. Thus, if you transplant tumours from one animal to another of the same species (usually mice) generally they don’t grow – in much the same way that transplanted organs (livers, etc.) are rejected by the recipient’s immune system. Transplant rejection occurs because the body mounts an immune response to the foreign (i.e. ‘non-self’) organ: transplantation works when that is reduced by matching donor to recipient as closely as possible and combining that with immunosuppressant drugs.

But here’s an obvious thought: if tumours transferred between animals don’t grow, their immune systems must be doing a pretty good job of recognizing them as ‘non-self’ and killing them off. If that’s true, how about trying to boost the immune response in cancer patients as a therapeutic strategy? It’s such a good idea it’s become the trendiest thing in cancer science, the field being known as immunotherapy.

Immunotherapy

The aim is to give a patient’s immune response a helping hand so it can kill their tumours. The stars of the show are a subset of white blood cells called T lymphocytes: that’s because some of them have the power to kill – they’re ‘cytotoxic T cells’. So the simple plan is to boost either the number or the efficiency of these tumour-killing T cells. The story is complicated by there being lots of sub-types of T cells – most notably T Helper cells (that do what their name suggests: activate cytotoxic T cells) and Suppressor T cells that shut down immune responses.

To get the hang of immunotherapy we need only focus on ways of boosting T Helpers but in passing we can hardly avoid asking “why so complicated?” Well, the immune system has evolved on a tight-rope, trying on the one hand to kill invading organisms whilst, on the other, leaving the cells and tissues of the host untouched. It works amazingly well but it can fall off both ways when either it’s overcome by the genomic gymnastics of cancer or when it exceeds its remit and causes auto-immune diseases – things like type 1 diabetes in which the immune system destroys the cells in the pancreas that make insulin.

Shifting the balance

We’ve seen that T cells (of all varieties) are among the ‘groupies’ attracted to the scene of growing solid tumours (in Cooperative Cancer Groupies and Trouble With The Neighbours) and so the name of the game is how to tweak the balance in that environment towards more efficient tumour cell killing.

Broadly speaking, there are two forms of cancer immunotherapy. In one T cells are removed from the patient, grown to large numbers and then put back into the circulation – called ‘adoptive cell therapy’, we’ll come to it in Part 2. The more widespread approach, sometimes called ‘checkpoint blockade’, uses agents that block inhibitory pathways switched on by tumours – in effect releasing molecular brakes that prevent T cell hyperactivity and autoimmunity. So ‘checkpoint blockade’ is a systemic method – drugs are administered that diffuse throughout the body to find their targets, whereas next time we’ll be talking about ‘personalized medicine’ – using the patient’s own cells to fight his cancer.

There’s one further method – viral immunotherapy – which I wasn’t going to mention but has been in the news lately to the extent that I feel obliged to make a trio with “Blowing Up Cancer” to follow Parts 1 & 2.

There’s nothing new about this general idea. Over 100 years ago the New York surgeon William Coley noticed that occasionally tumours disappeared when patients accidentally picked up post-operative bacterial infections and, from bugs grown in the lab, he made extracts that, injected into solid tumours, caused about one in ten of them to regress, with some patients remaining well for many years thereafter.

A new era

Even so, it took until 1996 before it was shown that blocking an inhibitory signal could unleash the tumour killing power of T cells in mice and it was not until 2011 that the first such agent was approved by the U.S. Food and Drug Administration for treating melanoma. In part the delay was due to the ‘agent’ being an antibody and the time taken to develop ‘humanized’ versions thereof. Antibodies (aka immunoglobulins) are large, Y-shaped molecules made by B lymphocytes that bind with high specificity to target molecules – antigens – humanized forms being engineered so that they are made almost entirely of the human protein sequence and therefore do not provoke an immune response.

92 FigCheckpoint Blockade Activates Anti-Tumour Immunity

Interactions between Receptors A and a suppress T cell activity. Antibodies to these receptors block this signal and restore immune activity against tumour cells.

Unblocking the block

We picture the tumour microenvironment as a congregation of various cell types with chemical messengers whizzing to and fro between them. In addition, some protein (messenger) receptors on cell surfaces talk to each other. The receptors themselves become messengers thus drawing the cells together – essential to bring killer cells into contact with their target. You can think of all these protein-protein interactions as keys inserting into locks or as molecular handshakes – a coming together that passes on information. Antibodies come into their own because they bind to their targets just as avidly as the normal signaling molecules – so they’re great message disruptors.

The sketch shows in principle how this works for two interacting receptors, A and a. The arrival of a specific antibody (anti-A or anti-a) puts a stop to the conversation – and if the upshot of the chat was to decrease the immune response, bingo, we have it! Targeting a regulatory pathway with an antibody enhances anti-tumour responses.

Putting names to targets, CTLA-4 and PD-1 are two key cell-surface receptors that, when engaged, trigger inhibitory pathways and dampen T-cell activity. Antibodies to these (ipilimumab v. CTLA-4; pembrolizumab and nivolumab v. PD-1) have undergone a number of clinical trials and the two in combination have given significant responses, notably for melanoma. So complex is immune response control that it presents many targets for manipulation and a dozen or so agents (mostly antibodies) are now in various clinical trials.

Déjà vu

So the era of immunotherapy has well and truly arrived but, as ever with cancer, it is not quite time to break open the champagne and put our feet up. Whilst combinations of antibodies have given sustained responses, with some patients remaining disease-free for many years, at the moment immunotherapy has only been shown to work in subsets of cancers and even then only a small fraction (about 25%) of patients respond. My correspondent Dr. Markus Hartmann has pointed out that the relatively limited improvements in survival rates following immunotherapy might be significantly enhanced if we took into account the specific genetic background of patients and determined which genes of interest are expressed or switched off. This information should reveal why some patients benefit from immunotherapy whilst others with clinically similar disease do not.

The challenge, therefore, is to characterise individual tumours and their supporting bretheren in terms of the cell types and messengers involved so that the optimal targets can be selected – and, of course, to make the necessary agents. It’s a tough ask, as the sporting fraternity might put it, but that’s what science is about so onwards and upwards with William Coley’s words of 105 years ago writ large on the lab notice board: “That only a few instead of the majority showed such brilliant results did not cause me to abandon the method, but only stimulated me to more earnest search for further improvements in the method.”

I’m grateful to Dr. Markus Hartmann  (Twitter: @markus2910) for constructive comments about this post.

References

Coley, W. B. (1910). The Treatment of Inoperable Sarcoma by Bacterial Toxins (the Mixed Toxins of the Streptococcus erysipelas and the Bacillus prodigiosus). Proceedings of the Royal Society of Medicine  3, 1-48.

Twyman-Saint Victor, C. et al. (2015). Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature 520, 373–377.

Wolchok, J.D. et al. (2013). Nivolumab plus Ipilimumab in Advanced Melanoma. N. Eng. J. Med., 369, 122-133.

Getting your DNA in a twist

I have a good friend who has just emerged triumphant from a run-in with bowel cancer – she’s in complete remission! Almost as wonderful is the fact that colliding with cancer has converted her from a genuine non-scientist to one who devours biology like fish and chip suppers. Spotting a recent volley of media items about four-stranded ‘quadruple helix’ DNA in human cells, she was on Twitter in a flash: “Does this mean that people with cancer have lots more quadruplex DNA than normal?” As she knows I can’t stand the Tweet cult she was probably amazed to get a reply: short answer: “No.”

But as ever in science, there’s a long(er) response. So, if you’re interested in the gyrations and gymnastics of which your genetic code is capable, read on …

The DNA double helix

The DNA double helix

Beautiful DNA

As you know, DNA comes as a double helix – a 2-chain spiral of small units (called nucleotides) that stick together (the units contain bases, so they’re ‘base-paired’). The oft-reproduced double helix image is beautiful because it’s a repetitive structure and you can easily see how it can be ‘unzipped’ so that each half can be used as a template to make a copy and regions can be ‘read’ to make RNA and proteins – though it was really designed to enable biologists to make endless unzipping jokes about genes and jeans.

The two DNA molecules of the helix stick together because of a balance between three forces: (1) weak electronic attraction between some atoms in the bases (called hydrogen bonds), (2) a sort of glueyness between the bases because their chemical structure means they don’t like water much and they’d rather snuggle up together, and (3) a repulsion between the chains because of the repeated phosphate groups all the way along the backbone (these carry an electric charge and likes repel, as we know).

Ugly DNA

But with all these attractions and repulsions you might think there would be lots of ways nucleic acids could get tangled up with each other – and there is. So the common form of double helix (the beautiful shape) is B-DNA but there’s also A-DNA, C-DNA and Z-DNA. If you just change the conditions a bit (pinch of salt or whatever) you can tweak the interactions so bits of the bases that don’t interact in B-DNA will do so to give a slightly different shape (usually a bit distorted – ugly). As you can see from the structure, Z-DNA is more Homer Simpson than Watson and Crick.

B- and Z-DNA

B- and Z-DNA

B-DNA and Z-DNA

We can’t reproduce the environment of DNA in the nucleus so we don’t really have a clue but the betting is that short bits of DNA jink in and out of these odd structural formations – just as part of the continuous flexing of the molecules. There’s also a couple of other things that can happen that have been known for a long time – again just dependent on the precise conditions in which a piece of DNA finds itself.

Sexy modelling

The first is a variant on the hydrogen bonds that form between bases. One way to think of this is to imagine two circles of five people, each ring holding hands and facing outwards. Each person is an atom in the bases of DNA. Let’s think of base pairing as the two nearest in the circles getting close enough to kiss. That’s one hydrogen bond. But, of course, the two other pairs on either side will now be quite close: if one of them also manages to kiss (tongues may be used) now we have two hydrogen bonds – which is what holds the bases A and T together. But suppose that the pair on the other side (who must also be quite close with all this adjacent necking going on) decide they really fancy joining in and are so excited that they twist the circle out of shape to do so. That this can happen has been known for years (it’s called Hoogsteen base pairing after the voyeur what spotted it) and when it does it can distort the helix enough for a third DNA strand to wrap round the original two – so you get triple-stranded DNA.

A sexual need

Similarly, if you tweak the conditions you can get four strands of DNA to come together and indeed we’ve known for yonks that happens naturally during recombination (that’s when genetic material gets swapped between Mum and Dad chromosomes – the reason for sex). When that happens you can think of four DNA strands forming a cross, each quadrant contains DNA from one strand of a chromosome, base-paired to that in the next quadrant – which is how bits get swapped around.

Non-sexual hugging?

So there’s nothing new about odd DNA shapes but what has made the news is that for the first time, rather than looking at what can be made to happen in a test tube, Shankar Balasubramanian and his pals have looked in whole cells. To do this they made an antibody that sticks only to ‘quadruple helix’ DNA structures – G-quadruplexes. The upshot is that they detected quadruplexes scattered throughout chromosomes and they see more in cells that are rapidly dividing than in ones that are just sitting there (they looked in some cancer cells in culture that do divide quite rapidly – but bear in mind that in tumours cells aren’t diving all that fast). So the inference is that they might form as part of DNA replication and, if you can target them by their antibody, maybe you could do something similar with a drug that would stop cells dividing. And if you could target that to cancer cells you could stop them in their tracks.

And the catch …

Simple. But there are some problems. It’s possible the antibody helps the quadruplexes to form – so it could even be a cunning artifact. But if we assume it isn’t – then we come face to face with a really big problem. There are zillions of ways you can kill cancer cells. The difficulty is that there isn’t one that selects cancer cells from normals. It may be possible (though it’s not evident how) to target quadruplexes and block cell division – but there are lots of cells that we need to divide rapidly just to keep us going – and, if quadruplexes are real, presumably they have ’em. So non-specific killing is probably not a good idea. Twas ever thus.

References

Biffi, G., Tannahill, D., McCafferty, J. and Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry published online: 20 January 2013 | doi: 10.1038/nchem.1548

http://www.cam.ac.uk/research/news/four-stranded-quadruple-helix-dna-structure-proven-to-exist-in-human-cells/