Starting a Research Group

It’s been a while since I last posted on here; over 3 years in fact. The original aim of this blog was to try and summarise some of our recently-published work without the scientific jargon that not only makes these papers incomprehensible to most people, but has the added disadvantage of putting them off what might (or might not!) be an important topic. This was exemplified recently when one of the papers from our lab was published in the scientific Journal Cancer Research. I asked my family how many words into the abstract they could get before getting lost. I lost them all on the third word - ‘endogenous’. This problem was magnified by the fact that the title made no sense to them either. So far, not so good. So I’m going to try a little harder to find the time to post here again in order to explain the science that we do and why I think it’s important.

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The Molecular Imaging Lab at King’s College London

Why haven’t you posted anything for ages I hear no one ask? Well, for the last ~5 years I have been been busy starting my own research Group. I say my own, but it’s not my own as it’s jointly owned by the very talented scientists that I get to work with every day (see pic). Together, we are part of a far larger Department at King’s where collaboration is deeply ingrained and the boundaries of each Group are blurred. But it’s taken me nearly 5 years to get to a point where I can reflect on this sometimes difficult process, which has seen me move Universities and start all over again. I thought I’d write about what it’s like to start a research Group, the errors that I have made, and the joys of being able to perform the science that really interests me.

The five stages of grief

Don’t worry, this story has a happy ending! But starting a Group is not easy. Up until this point we have been trained to be very good scientists but no thought has been put into what it takes to be a good manager, how to handle finances or handle high-level project management. So doing these things for the first time can often feel like a big shock to the system and has probably been the hardest thing I’ve done in my career to-date. I forgot who told me this, but they compared starting your own lab to the five stages of grief, which on refection is a great analogy. It’s reproduced below. I should stress that this is in no-way the process that everyone goes through, but it resonated with me. Of course it goes without saying that this is just a way of illustrating the different emotions I went though over the past five years and in no way can be compared to the tragedy of losing someone important to you.

1. Denial

It all starts with money. Actually, that’s not strictly true - it starts with an idea, but that idea will die unless you can get a research grant to fund people and materials to enact that idea. I was fortunate enough to be awarded a Wellcome Trust & Royal Society Sir Henry Dale Fellowship that enabled me to do just that. It’s hard to describe the joy of receiving the news that my Fellowship had been funded. But after that initial euphoria dies down you’re left with the task of having to hire for the very first time, find space to perform experiments, get quotes for everything from the most important piece of equipment to a basic fridge. The list is long; sometimes exhaustingly so, which can lead to avoidance of the issues, procrastination (see twitter and this website) and an overwhelming sense of the magnitude of the task. Luckily I had thought a lot about what I would like my Group to look like, but there’s no substitution for actually going through this experience yourself. In many way it’s like becoming a parent for the first time - no matter how many people you talk to on the subject, nothing prepares you for the real thing.

2. Anger

Anger is probably a little extreme, but once you’ve got everything together and you’re ready to go you’d think that this would be it and everything would fall into place. Unfortunately, everything takes longer than you first thought. From hiring the right person (sometimes this means having to readvertise), finding lab space that was initially promised to you which has now mysteriously disappeared, to the dawning realisation that politics plays a prominent role in science and you’re the bottom of the rung. It’s not anger, but more of a frustrating process of having to get all your ducks in a row. One of the most annoying things that’s immediately obvious is the fact that suddenly you have to prove yourself all over again. As if the past maybe 8 years or so was a combination of luck and your previous PI’s genius. Which I can’t rule out in my case!

3. Bargaining

You might have some equipment and if you’re lucky, a postdoc or grad student to micromanage (sorry, I mean mentor…), but quickly I realised that a great idea, willing hands and some pipettes is not enough and you really need some outside help to sustain you and your research in the early days. There are many colleagues over the last few years that have helped me with no expectation of the help being repaid, be it reading drafts of grants, acting as a sounding board, or having the patience to teach your student a new technique. However, further afield, forget it if you think people will magically know either who you are or will want to collaborate straight away. You have no track record as a PI, so it’s often seen as a risk. People are not knocking down your door to work with you. So how to get around this problem in the early days? Often it’s a case of bargaining - finding areas of mutual interest, supporting research that’s not directly related to your own programme and frequently exchanging important data and analysis for a name on the paper that is yet to be written.

4. Depression

Which is where the shock hits. Having pretty consistently published at least one first author paper a year during my postdocs, the reality is that even with many more people working on the same idea or project, it takes over double the amount of time to get your first paper published. In fact, it took us more than three years before we started sending our paper out for rejection. I mean, publication. This first paper is often your baby, so you know exactly what form it will take. But there’s a lot of training to do. And now you’re needed to teach, be on interview panels, speak at conferences, complete all the admin the U.K.’s University system seems to thrive on. Things take their time in the lab. You might not have the same resources you were used to either. This is all against the background of some sort of ticking clock when you know you have to either deliver or be out of a job, ~10-15 years after starting and with no hope of surviving in the ‘real world’. I was really feeling the pressure three years in and I think not only I, but my Team suffered because of the worry that surrounded the fixed-term contract that I was on. This is maybe a topic for another day, but Universities really need to look hard at how they support their Research Fellows. Put your money where your mouth is and stop taking advantage of highly-skilled Young Investigators. Sorry, I’ve gone back to step 2. Back on topic, once the first paper from your Group is ready, there is so much excitement surrounding the paper and the endless possibilities. Why wouldn’t Nature take it?! I mean, we’ve all seen worse published there. You may be lucky, but we aimed high and were persistent. So were the desk rejections. The few months before our first work was published in Cancer Research was a tough time.

5. Acceptance

But then things start clicking into place. You find your rhythm managing people, some grants are funded, and a lot aren’t. But you start getting used to rejection as part of the everyday way of things. It’s a necessary, but unfortunate side product of running your own Group. From one idea new data spawns many more; made all the more joyous when these come from other members of the Team. Publishing certainly helps the confidence and the realisation that running a research lab is a good fit for you. Acceptance that you are learning and won’t get everything right all the time also comes. I used to spend a lot of time worrying when I got things wrong, but now I try and spend the time planning for that difficult conversation or learning from past mistakes. Acceptance is also about admitting when things aren’t quite right. This realisation was the catalyst for me moving from UCL down the road to King’s.

There are some beautiful moments that come from running a lab. Some of my highlights include seeing my former PhD supervisor frantically scribbling down notes during one of my talks, the excitement of my PhD student hearing her project described for the first time, and being invited by your long-time friends to see their labs and speak at their seminars. It’s not for everyone, but it’s certainly for me.

A beginner’s guide to imaging in vivo tumour biochemistry

Here we discuss how imaging can be used to understand biochemical processes in living subjects without the need for invasive procedures. It’s written for a non-specialist audience and is a good starting point to learn more about the subject.

The article was originally published in The Biochemist and is authored by Hannah Greenwood, Richard Edwards and Tim Witney from the Molecular Imaging Lab.

Using antimatter to look at cancer

Cancer can be defined as the uncontrolled proliferation of cells in the body that arise from genetic mutation. Cancer is a devastating disease, accounting for more than a quarter of all deaths in the UK (Cancer Research UK, https://www.cancerresearchuk.org/health-professional/cancer-statistics-for-the-uk, accessed May 2019). Over the last couple of decades, a detailed understanding of the molecular pathways associated with cancer growth, progression and metastases has led to the development of new and improved treatments that target these pathways. For example, the identification of the HER2 receptor, with the resulting drug, Herceptin, has produced long-term survival benefits in many women with HER2-positive breast cancer. We are able to characterise, measure and visualise these important biochemical processes in a living system using a powerful medical imaging technique called positron emission tomography (PET). Unlike other medical imaging technologies, such as computed tomography or magnetic resonance imaging, which provide detailed anatomical information, PET can reveal the functional processes that drive disease without the need for surgical intervention. In this article, we will discuss the steps involved in developing ‘smart’ imaging probes to visualise in vivo tumour biochemistry with PET. 

Cancer is a heterogenous disease, with multiple phenotypes observed both between different metastatic sites and within the primary tumour itself. Tumour heterogeneity has wide-spread implications for patient diagnosis, treatment selection, and ultimately how these tumours respond to therapy. Diagnosis is traditionally confirmed by biopsy following referral by a GP, often after the detection of a suspicious growth or the onset of symptoms. Biopsies are an invasive procedure in which a small region of the suspected lesion is surgically excised and examined under a microscope to assess the presence (or absence) of malignant cells. Tumour biopsies, however, hold a number of limitations: As only a small region of the tumour is sampled, a single biopsy may not capture the whole picture of the disease itself. In extreme cases, inadequate sampling can lead to the misdiagnosis and wrongful treatment of patients. In addition, acquiring tumour biopsies is an invasive procedure, and in some cases a biopsy cannot be obtained at all due to the location of the mass. By comparison, PET imaging can provide doctors with whole-body images of cancer biochemistry. This allows the assessment of both primary tumours and distant metastases. Seeing ‘the whole picture’ allows for more accurate diagnosis and improved patient care.

PET imaging works through the indirect detection of an antimatter particle called a positron, emitted from an unstable atom, known as a radionuclide. These radioactive atoms can be attached to a molecule of biological interest to produce a radiolabelled molecule known as a radiotracer. Following its intravenous injection into a subject, the radiotracer accumulates at the selected biological target of interest and is detected by a PET scanner. A detailed description of this process is described in Figure 1.

Figure 1. Preclinical PET imaging. Prior to acquiring a PET image, a radiotracer is injected into the subject (1). Radiotracers are made by attaching a radioactive atom (radionuclide) to a scaffold that targets a biological process of interest. As the unstable radioactive atom decays it emits a positron (the antimatter equivalent of an electron), which travels a shorts distance (~1 mm) before colliding with a near-by electron (2). Upon collision, these two particles undergo a process called ‘annihilation’, releasing two gamma rays at ~180 degrees to one another. The detection of these gamma rays by a PET scanner allows for the origin of annihilation to be calculated, subsequently providing the precise location of the radiotracer. Computer processing enables multiple annihilation events to be processed, building a 3D ‘map’ of radioactivity inside the body – the PET image (3).

Designing smart imaging probes

If properly designed, a radiotracer can allow us to visualise important molecular processes relating to cancer. Before designing the radiotracer itself, an informative biological target to image must be selected. This biological target is called a ‘biomarker’. A biomarker can be an indicator of normal biological processes, disease processes or responses to an intervention, such as drug treatment. Important examples of biomarkers relating to PET imaging include receptors, enzymes, proteins, and transporters. The data obtained by the measurement of a well-selected biomarker can then be used to answer important clinical or fundamental question relating to cancer biology.

In a clinical setting, imaging will look to address a question regarding how to treat a patient. This could be ‘Does this patient have cancer?’, ‘How is the patient’s cancer responding to their therapy?’, ‘Will the patient’s cancer respond to this type of therapy?’ or even ‘What is the cancer phenotype?’. The different biomarkers exhibited by cancer cells can be targeted by PET probes to achieve selective uptake and accumulation in specific cancer types to answer these fundamental questions. This accurate characterisation of the cancer allows for treatments to be selected for individual patients rather than the population as a whole in a process known as precision medicine.

Successful visualisation of the biomarker relies on ‘contrast’. Firstly, this means that the target must be differentially expressed in cancer over that of the background tissue, with any background uptake of the radiotracer clearing within the lifetime of the radioactive atom. Without this clearance, contrast cannot be achieved and visualisation of this biomarker will be not be possible. Finally, for the visualisation to be informative, the radiotracer must be delivered to, and specifically interact with its biological target. This requires appropriate design and application of the radiotracer.

An excellent example of a PET radiotracer for cancer imaging is [18F]FDG, a glucose analogue which is taken into cells by glucose transporters and phosphorylated by the enzyme hexokinase (Figure 2A). The replacement of a hydroxyl group for the radioactive fluorine isotope means that after intracellular phosphorylation the sugar becomes trapped as due to its altered structure it cannot be further metabolised. The increased glucose metabolism exhibited by cancer cells means that [18F]FDG is preferentially taken up, allowing for high contrast imaging of tumours. This is a perfect example of how a radiotracer can be used to measure altered cancer biochemistry, which may inform treatment decisions. [18F]FDG is now used in most modern hospitals for disease staging and confirmation of primary diagnoses. As we move towards more personalised medicine regimens, however, there is a requirement for probes not only to detect cancer but to distinguish cancers of a specific phenotype or aggressiveness. This requires the development of new, ‘smart’ imaging probes. In the design of these informative probes, a number of considerations must be made:

1. Where is the biological process occurring?

In order to image a biochemical process, the probe needs to be able to interact with the system under investigation. Delivering molecules and biological scaffolds to where you want them in the body is far from trivial. It is a problem that drug companies and their medicinal chemistry teams spend billions of pounds trying to solve. The same problems need to be addressed in PET imaging. Firstly, the radiotracer needs to get to the site of disease. This can be tricky as the complex in vivo environment means that many compounds are quickly cleared (removed from circulation) or broken down in the body. The compound may also need to cross physical barriers such as the blood brain barrier or a cell membrane. Correct selection and/or design of the radioactive probe is key to overcome these issues.  As a result, the design of the radiotracer will vary considerably depending on the location of your biomarker, not only within the body, but at the cellular level. The properties of a radiotracer designed for an extracellular target can vary dramatically from a radiotracer imaging an intracellular or transmembrane target, as will be discussed below.

2. What type of scaffold will I use?

Scaffolds that can be radiolabelled for the production of PET radiotracers include small molecules, peptides, proteins and antibodies. Each of these scaffolds can target biomarkers to produce an informative image.

Small molecules, such as inhibitors or substrates can target intracellular proteins or enzymes. The intracellular location of the target means that the radiolabelled probe must cross the cell membrane. This may require the design and tuning of a small molecule’s properties, whilst radiolabelled endogenous species, such as sugars and amino acids, may have mechanisms for cell internalisation, often via membrane transporters. The probe can accumulate in the cell by binding to its recognition site (Figure 2B). Alternatively, a substrate-based probe can undergo a modification that leads to its ‘trapping’ in the cell. This ‘trapping’ can be due to alterations leading to an inability to cross the cell membrane once it enters the cell (e.g. phosphorylation of FDG - shown in Figure 2A) or binding at another intracellular site. Small molecule cell transporter substrates can also be radiolabelled to detect differences in cell transporter expression.

Peptides, proteins and antibodies are typically used to detect extracellular biomarkers. Advantageously, any issues regarding the cell membrane permeability of these scaffolds are negated for extracellular biomarkers. Radiolabelled versions of these scaffolds bind to specific receptors on the cell surface. This allows visualisation of tumours exhibiting increased receptor expression compared to healthy cells (Figure 2C). This can inform a clinician if a patient is likely to respond to therapy targeting this particular cancer type, e.g. HER2-expressing tumours.

Figure 2. Different radiolabelled scaffolds and their applications. A) [18F]FDG is transported in to cells where it accumulates due to metabolic trapping subsequent to phosphorylation by hexokinase. B) A radiolabelled drug molecule crosses the cellular membrane and binds to its intracellular protein target. C) Radiolabelled antibodies bind to the extracellular domain of specific receptors.

Synthesis of smart imaging probes

The synthesis of radioactive molecules (termed radiochemistry) has unique challenges, most prevalent of which is the synthesis and purification of the radiotracer within the time constraints of the decaying radionuclide. Production and purification should normally be completed within 2 half-lives of the radioactive atom (before 75% of the starting radioactivity has decayed) so there is enough radioactive material left to enable reliable imaging of the disease. Synthesis is further complicated when using large amounts of radioactivity. While the opening credits of ‘The Simpsons’ may suggest that a biohazard suit and a pair of tongs are suitable protection for handling radioactive material, this certainly isn’t the case for PET radionuclides! The gamma radiation produced is highly penetrating and is what permits the deep tissue visualisation of PET probes. This means that standard organic chemistry procedures are impossible, or certainly not advisable! To overcome these problems, robotic systems have been developed to perform the desired manipulations in an automated fashion. These systems can then be placed in a heavily shielded compartment known as a ‘hot cell’ and controlled remotely.

The radiolabelling process depends on the scaffold chosen and the radioactive atom (radionuclide) selected. The half-life of your radionuclide should match that of the biological process you’re interested in visualising – you don’t want all of your radiotracer to decay before the meaningful data is acquired. For example, the radionuclide fluorine-18 (half-life of about 2 hours) is very popular for labelling small drug molecules, as these compounds are rapidly removed from the blood and excreted. This allows contrast, and therefore visualisation, of your target a short time after injection. Labelling an antibody with fluorine-18 would be impractical, however, as the radiotracer would completely decay before its removal from the blood, which can take many days. For visualisation of a labelled antibody’s target, better contrast is achieved much later. Therefore, a radionuclide with a longer half-life is generally used, such as zirconium-89 (half-life of about 3 days), so that contrast can be obtained. Once the radionuclide and the scaffold being labelled have been selected, a number of strategies are available to incorporate or attach the radioactive atom.

One approach is to produce a direct radioactive equivalent of your molecule of interest (Figure 3A). Advances in both radiochemistry and the robotic platforms used to perform the manipulations, have led to remarkable advances in our ability to incorporate radioactive atoms into structures of increasingly complexity. The radiolabelling can be performed using the radionuclide itself (e.g. fluorine-18 fluoride for fluorination) or by using radioactive reagents or building blocks (e.g. carbon-11 methyliodide for methylation) to synthesise the molecule. Alternatively, small molecules or peptides can have small radioactive units known as ‘prosthetic groups’ attached to them to facilitate radiolabelling where direct substitution of a non-radioactive atom for one that’s radioactive is not possible (Figure 3B). Larger species such as antibodies are amenable to more considerable alteration, whilst maintaining their original biological behaviour. This allows for functionalisation with larger structures such as chelators for coordinating radioactive metals including zirconium-89 or copper-64 (Figure 3C).

Figure 3. Different strategies for radiolabelling

As our understanding of how to deliver a radiotracer to its desired target progresses, radiotracer design can improve. In most cases, success will not be achieved at the first attempt. Novel radiotracers must be validated biologically (as discussed below). If the in vitro or in vivo studies show that the compound is not fulfilling the requirements of an informative radiotracer, the design and synthesis processes must start again using the new information acquired.

From radiosynthesis to imaging

Following the identification of an appropriate biomarker and development of the complementary imaging agent, cell-based validation must be completed to confirm the specificity of a probe to its target. Here, immortal cancer cell lines are incubated with the radioactive probe over a defined period of time. Scientists can then compare radiotracer accumulation or binding to cell lines with altered characteristics, for example, a drug sensitive vs. a drug resistant cell line, or cells expressing HER2 and those that do not. To check the selectivity of a probe to its target, blocking assays, competition assays or gene knock-down models are often used. Blocking and competition assays are used to prevent target-probe interactions. If the probe is specific to the biochemical target that has been blocked, no radioactivity should be detected in the cell. Knock-down models work in a similar way. Rather than blocking the target, the target gene and therefore biomarker is removed completely. If the novel probe is specific for the gene that has been knocked-out, there should be negligible radiotracer binding – demonstrating selectivity of the probe to the specific biomarker of interest. These initial experiments are necessary before the probe can be translated into complex living systems. Redesign of the imaging probe is required if non-specific binding is observed in these relatively simple models.

If the new radiotracer is shown to specifically interact with the biomarker of interest in cell culture-based experiments, validation then progresses to small animal research and in vivo (live animal) imaging. Tracking of the probe in healthy rodent models is performed prior to disease models. Experiments in healthy mice are important to understand the healthy tissue clearance and subsequent excretion route of the novel radiotracer. The two main excretion routes are via the renal system (kidneys and urine) and hepatobiliary system (liver and the intestines). The excretion route of the probe will determine which cancer types can be imaged. For example, a radiotracer that is cleared through the hepatobiliary system would not be appropriate to examine liver cancer due the high background signal in healthy liver tissue.

A crucial part of radiotracer assessment in vivo is the selection of clinically-relevant cancer models. The appropriate model could be a subcutaneously implanted patient-derived tumour xenograft using tissue taken directly from a cancer patient; an orthotopic tumour model, where tumour cell lines are grown in the tissue from which they originate; or a spontaneously-arising tumour generated in a genetically modified mouse, to name a few. Preclinical research provides vital information regarding a radiotracer’s binding to the target of interest, as well as information on how it is broken down. Dedicated small animal PET imaging scanners offer a platform for whole body, dynamic evaluation of these probes, often combined with computed tomography (CT) for anatomical reference.  If the probe has been successfully delivered to its target, a ‘hotspot’ of radioactivity will be seen in the reconstructed PET image (Figure 4). The reconstructed PET image also allows scientists to observe the stability of the radiotracer. For example, when using a probe labelled with fluorine-18, if radioactive signal is seen in the bone, this is a sign of defluorination, where the radioisotope has become detached from its scaffold.

It is important to understand the in vivo binding properties, toxicity and radioactive dose delivery of novel imaging agents for Medicines and Healthcare products Regulatory Agency (MHRA) approval, prior to testing in humans. These tests are imperative for the complete validation of novel imaging agents but it must be noted that cell culture test or in vivo model will never replicate exactly what will be seen in humans. Clinical trials are critical in evaluating the efficacy of novel imaging agents in the diagnosis and management of diseases.

Figure 4. Preclinical versus human PET/CT imaging. Preclinical PET/CT allows whole-body visualisation of the radiotracer’s biodistribution, dynamically in small rodent models. In current clinical PET/CT systems, whole-body imaging is not possible as the whole body of the patient can not fit within the axial field of view of the scanner (usually 22 cm). This means only static scans, looking at the radiotracer distribution at a specific time, can be viewed. The clinical PET/CT image shown is a composite image from at least 2 or more bed positions. Developments in technology (and a lot of money) have led to the first total-body PET scanner – the EXPLORER.

The future of PET – antimatter matters

PET has advanced drastically from a technique used to simply visualise cancer lesions. One notable success is the emerging field of theranostics, which merges diagnostics and therapy into a single technique. As in conventional PET imaging, the first part of theranostic imaging is the diagnosis of a disease. A specially selected scaffold tagged with a PET radionuclide is injected into a patent where it will accumulate at the target of interest (if present). This accumulation of the radioactive probe will then be detecting during a PET scan. If there is high accumulation of the probe, the next part of theranostic imaging is to deliver a targeted amount of therapy to the patient. For this, the same scaffold is used; however, the gamma emitting radionuclide is replaced with a corresponding isotope that emits alpha particles. Alpha particles are highly charged and heavy atoms, and as a result they lose their energy in a very short distance, damaging the surrounding cells by causing double stand DNA breaks, for example. Specific delivery of these alpha particles to a cancerous lesion therefore gives a localised dose of therapy, which has been shown to be a highly effective treatment. This technique enables both selective imaging with the PET analogue, followed by therapy with the therapeutic equivalent. The most commonly used theronostic pair is technicium-99 and iodine-131 for the management of thyroid cancer.  As well as thyroid cancer, theranostics has been extremely successful in the treatment of patients with neuroendocrine tumours and prostate cancer.

Excitingly, advances in scanner engineering have led to the development of the world’s first total-body PET scanner – the EXPLORER (Figure 4). The EXPLORER has an axial imaging field of view of 194 cm, almost 10-fold greater than a conventional PET/CT, allowing the entire body to be imaged at the same time. This milestone will provide previously unseen information on the whole-body dynamics of PET radiotracers. Lower doses, faster scanning and the ability to image at later time points will also be beneficial for patients and clinicians alike. The increased sensitivity that whole-body PET can offer will be particularly advantageous in cancer imaging, allowing the detection of small lesions that previously would go undetected.

Figure 5. The development of a novel PET imaging probe. Following the selection of a suitable biomarker, radiolabelling of the appropriate scaffold is performed. The radiotracer is first validated in cells to examine specificity and selectivity to the biomarker, before being injected into a mouse model for preclinical in vivo PET imaging. If the results of these tests show promising results, testing of the radiotracer moves into human clinical trials, following MHRA approval.

Conclusions

Imaging tumour biochemistry in vivo is a complex process that requires a collaborative approach from a multidisciplinary research team. We have discussed the processes that are involved in selecting a biochemical target to image, synthesising the probe and testing the radiotracer both in cells and in vivo prior to clinical translation (Figure 5). Non-invasive imaging of tumour biochemistry using new, smart imaging tools, places imaging scientists in a position to understand the mechanisms that drive cancer progression and the ability to treat the tumour whilst leaving healthy organs unaffected. The field of PET must strive to meet the demands of the medical community by delivering probes that can provide meaningful patient information, thereby improving the management and outcome of this devastating disease.

Further Reading

• James, M. L. and Gambhir, S. S. (2012) A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications. Physiol. Rev. 92, 897–965. doi: 10.1152/physrev.00049.2010 https://www.physiology.org/doi/full/10.1152/physrev.00049.2010

• Witney, T. H. et al. (2015) PET imaging of tumor glycolysis downstream of hexokinase through noninvasive measurement of pyruvate kinase M2. Sci. Transl. Med. 7, 310ra169-310ra169. doi: 10.1126/scitranslmed.aac6117 https://stm.sciencemag.org/content/7/310/310ra169

• Badawi, R. D. et al. (2019) First Human Imaging Studies with the EXPLORER Total-Body PET Scanner*. J. Nucl. Med. 60, 299–303. doi: 10.2967/jnumed.119.226498 http://jnm.snmjournals.org/content/60/3/299.long

• van der Born, D. et al. (2017) Fluorine-18 labelled building blocks for PET tracer synthesis. Chem. Soc. Rev. 46, 4709–4773. doi: 10.1039/c6cs00492j https://pubs.rsc.org/en/content/articlehtml/2017/cs/c6cs00492j

• Kostelnik, T. I. and Orvig, C. (2019) Radioactive Main Group and Rare Earth Metals for Imaging and Therapy. Chem. Rev. 119, 902–956. doi: 10.1021/acs.chemrev.8b00294 https://pubs.acs.org/doi/10.1021/acs.chemrev.8b00294

Flexibility is a killer

I'm often asked why, despite many $/£ millions have been spent on cancer research, it seems like we're no closer to a cure. This is quite a difficult question to answer, because there are a number of factors that contribute to cancer being such a tough disease to treat. I should point out though, that some really great progress has been made in recent years, exemplified by breakthrough immunotherapy drugs, where our own immune system is trained to kill the cancer; or new combination drugs that target specific genetic mutations found in many cancers. Also, as Cancer Research UK's very informative website states, cancer survival rates have doubled in the last 40 years. But this really isn't good enough. There are a variety of reasons why we're struggling to do better, but in my mind there are two predominant reasons why cancer is such a moving target:

1. Each cancer is different and specific to each individual patient. Cancer results from problems arising in our own DNA, known as gene mutations. The problem is that for each person, these 'problems', or mutations, occur in different places in the DNA, affecting different genes. Each mutation really needs a different treatment, meaning that we need a whole library of drugs to be effective. There are efforts to try and tailor each treatment to individual patients, known as personalised, or precision medicine. 

2. Cancers are adaptable. By their very nature, they are designed to grow and outcompete normal healthy tissue that surround them. They co-opt new blood vessels to provide them with the nutrients they need, and can change their behaviour in order to survive, say drug treatment, or to enable them to grow in harsh environments. 

Over the past couple of years at Stanford University, I've worked on trying to better understand how this adaptability gives cancer an unhealthy advantage over normal cells, and the organs they colonise and take over. One element of flexibility that cancer cells possess is the ability to choose how they use the sugar they take up. Sugar, in the form of glucose, is needed for all cells in order to create energy that keeps the cell alive. To produce this energy, glucose is taken up by cells and converted to other molecules in a process called 'glycolysis'. When oxygen is present (the reason why we need to keep breathing!), these molecules are further modified and energy is produced. Cancer cells have generated a unique ability to put the brakes on this process. On the face of it, this seems like a bad idea, but what this means is that the molecules of glycolysis usually used for creating energy can now be used to make other things, such as new bits of cells. The result is that more cancer cells are produced. Cancer cells can quickly switch between these two states: 1) energy production; or 2) the creation of new building blocks to create more cancer cells. 

Fig. 1. PKM2 controls the balance between the production of new building blocks or energy in cancer cells. Here, PKM2 is represented by an orange sphere.  

We're now starting to understand what controls this flexibility inside cancer cells, and it's an enzyme called pyruvate kinase M2, or PKM2. PKM2 has been found in all cancer cells studied to-date, and acts as the master regulator of glycolysis. When the cancer cell requires energy, PKM2 binds to 3 other identical PKM2 enzymes, but when new building blocks are needed for the creation of new cells, PKM2 only binds to one other PKM2 enzyme, blocking energy production almost completely (Fig. 1). Most normal healthy cells lack PKM2, instead using an enzyme, PKM1, that always leads to energy production.

Fig. 2. DASA-23 binding to a tumour in the brain of a mouse, indicated by the arrow.

In the lab of Sanjiv Sam Gambhir at Stanford, my colleagues and I worked on ways to try and detect this cancer-related enzyme in animal models of human brain cancer. We reasoned that if we could detect PKM2, we may be able to detect the cancer itself. This would be massively helpful to the brain surgeon when they came to remove the brain tumour as they would know its precise location within the brain. This is important, as the surgeon doesn't want to take too much healthy tissue, which may result in unwanted side effects, or be too cautious and leave some of the tumour behind. We developed a new positron emission tomography imaging agent (see earlier posts for an overview of this technique) based on a known drug, DASA-23, that specifically targets PKM2. By tagging DASA-23 with radioactivity and injecting it into mice, we could see its precise location within the animal and how that changed over time. As shown in Fig. 2, the tagged DASA-23 honed precisely to the PKM2-containing brain tumours and not to the healthy surrounding brain tissue. Moreover, we showed that by adding another drug that targeted PKM2 we could stop DASA-23 binding to the tumour cells. This showed that the new drug was going to the right place, meaning that DASA-23 might be useful for the development of new PKM2-specific drugs. Although exciting, it should be pointed out that these are very early findings and a lot more research is needed before we will be able to use DASA-23 in hospitals around the country, but early tests in humans are planned for next year.

If you're interested, this work was published as the front cover of this week's Science Translational Medicine. 

Measuring fat breakdown to detect cancer

Last year I moved to the United States to take up a postdoctoral position at Stanford University in the lab of Prof. Sanjiv Sam Gambhir. I'm lucky enough to work in a very creative environment, nicely summed-up in this video. Living in the US however has brought some challenges I've been ill-equipped to deal with. Namely, how to avoid getting run over when crossing a road (walking seems to be prohibited here) and what to do to stop getting fat. High fructose corn syrup is added to pretty much everything and it costs nearly the same amount to eat out as to buy the constituent ingredients (eating out is cheap!). I don't stand much of a chance. Running across the road however may solve both issues.

Given my rapidly-expanding waistline, it seems pretty appropriate that my research during my last year at Imperial College London focused on measuring the breakdown of fat by cancer cells. As I mentioned in my last blog, cancer cells take up more glucose for energy production and storage. Additionally, tumours require increased levels of fat to make new cells and to create even more energy. The breakdown of fats at high rates to produce this energy sets cancer cells apart from most normal 'healthy' tissue. We have recently shown that by imaging fat breakdown, we can detect breast, prostate and brain cancer in preclinical (non-human) models, published this month in The Journal of Nuclear Medicine. This is important as existing techniques to identify and diagnose both brain and prostate cancer are not effective in all cases. Further tests, such as the one proposed in this research article, may provide additional information and eliminate the need for an invasive biopsy. By accurately detecting these cancers at an early stage, the chances of survival are greatly improved. We're still some way off evaluating this diagnostic test in patients, but I have high hopes for this new imaging technique, an example of which is shown below:

In other cool news this week, my academic mentor at Stanford, Prof. Sanjiv Sam Gambhir, is partnering with Google's secret research division, Google [x] - the division that's brought us Google Glass and those internet balloons. The idea behind this project, named Baseline, is to define and thoroughly characterise the genetic and molecular make-up of healthy adults (initially from 175 people, increasing to many thousands). By understanding the key features of good health, it's hoped that we may be better placed to understand and detect things that go wrong. Details of this project are light on the ground, but it's thought that some cool wearables, such as the 'smart contact lense', will be used to monitor those enrolled in the project 24/7. Let's hope this ambitious project results in a major scientific breakthrough.

Cancer's sweet tooth

Otto Warburg

One of the best things about being a scientist is discovering something no one has ever done or seen before. Whether it be the creation of a new man-made plastic, or the discovery of the Higgs boson, science is tirelessly expanding our collective knowledge. Sometimes however, we're so busy focusing on the horizon and the next scientific breakthrough that we forget to look over our shoulder and examine in sufficient detail what has come before.

Cancer detection through imaging

A German scientist, Otto Warburg made the discovery in the 1920s that cancer cells consume sugar in far greater amounts than normal healthy cells.  It's only recently though that we have started to use this discovery to our advantage.  By designing drugs that curtail this 'addiction' to sugar it is hoped that we can stop these cancer cells from growing. We also take advantage of cancer's sweet tooth during diagnosis. Following injection of a radioactive sugar into the bloodstream, clinicians can detect cancer using a scan that measures where that sugar is being used in the body.  An example is shown to the left, with the tumour indicated by the arrow.  It is now thought that cancer cells use the sugar to protect against harmful waste products, for energy, and to create building blocks to form new cells.

Following in the footsteps of Warburg, a team of French scientists made the discovery in the late 70s/early 80s that cancer cells save some of this sugar for a rainy day - when extra energy is needed, or to keep the cells alive when the supply of sugars from elsewhere runs out. This discovery is fascinating given that sugar stores are normally only found in the liver and muscle. Cancer cells that originate from say the breast or ovary seem to acquire the ability to store these sugars through, as of yet, unknown mechanisms. These findings have been largely ignored until now. In a research article published this month in Cancer Research, myself and my colleagues at Imperial College London further explore this phenomenon, some 30 years later.  We showed that cancer cells store more sugar when they stop growing and that we can detect these sugar stores through imaging. A picture of these sugar stores are shown below, indicated by the intense orange/yellow dots within the cancer cells. The identification of these stores has wider implications as cancer cells that grow more slowly are typically more resistant to traditional chemotherapy. It's hoped that this new imaging method might be able to detect these slower growing cells that we can then target with different drugs. Although this technique hasn't been tested in humans yet, we are hoping scans, similar to the one shown above, will be performed in the next few years.  There is also hope that this technique can be used to detect other sugar storage diseases such as diabetes.

Cancer's sugar stores

For more information, the research article, 'A Novel Radiotracer to Image Glycogen Metabolism in Tumors by Positron Emission Tomography' can be found here.