PhD – 21 months in 

Do you remember my optimistic blog post about finding my bearings in the lab after a month of the PhD? I also included pictures of a failed western blot and slightly crushed centrifuge tubes.

Well, twenty months later and I’m still making mistakes. Often they’re new and different mistakes, which could almost be exciting. But today I made the same mistake and lost a lot of plasmid-growing bacteria (bacteria I am using as work horses to produce specific DNA for me) in a centrifuge (which I subsequently cleaned!)…

Photographic evidence attached.

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Cancer Research UK – PhD Student Meeting

Cancer Research UK (CRUK) is the world’s largest independent cancer charity (according to Wikipedia)  and funds thousands of scientists across the UK. In the latest annual report they state that more than £340 million were spent on research. Take a look at the fancy infographic here to see a break-down of how that money was spent:

annual report

The reason for writing this post on CRUK is that today the charity held its first-ever first year PhD students’ meeting in London, at the Quaker Friends House near Euston station. The attendees came from all over the UK: from as far afield as Aberdeen and Manchester to Oxford and Cambridge and finally us lazy Londoners who could afford to get up later than on a normal lab day.

The main aims of the meeting were to get to know some of the people working at CRUK’s head office in London and how, in future, we might apply for their funding. One of the first things I learnt today was that CRUK has made four cancer types – brain, lung, pancreatic (!) and oesophageal – “strategic priorities”, because the survival rates for these are still low and lagging behind those of, say, breast and prostate cancer. We also heard, from the senior research funding manager, Richard Oakley, how CRUK spends its money and what we can and are meant to do to help. Among other things this involves wearing branded t-shirts and participating in fundraising events. So tomorrow morning I will wear this to run in the park in preparation for the 10 km Race for Life happening at the end of June – please feel free to fund me and/or the maybe pink team and/or join the run! [We can start a separate conversation on the topic of the martial language used by CRUK, and other charities, to help raise the money. N.B. The back of the t-shirt reads, “Ask me about my life-saving research.”]

cruk

Since doing a PhD is all about the learning experience, most of the morning was filled with one of three workshops on either a) assertiveness, b) time management, or c) having an effective working relationship with your supervisor. I chose the first option, and although some people (especially in my lab) will argue that it would be better if I were a bit more quiet on occasion, I thought it would be interesting to see what it could offer. The basic message was, of course, quite clear: effectively communicate your needs whilst appreciating other people’s needs. Easier said than done for sure. The only thing that helps is practising being in potentially awkward situations and putting oneself outside one’s comfort zone, which is where learning can happen. Possibly the most helpful information was to realise what isn’t assertive behaviour (e.g. being too passive, or too (indirectly) aggressive) and making sure to recognise those behaviours in oneself and learning to avoid them in future. We’ll see how that goes.

Over lunch we got to browse a few posters. I particularly enjoyed the ones on intestinal stem cells and a potential preventative treatment for breast cancer using the diabetes drug Metformin. And lastly, we were politely, with the help of beer and/or wine, coerced into networking…

A PhD Student for a Month

After the week of induction I have now spent three weeks in the lab. I have cells to look after, so that is most definitely a good thing. Mainly I am trying to figure out what kind of experiments to do that will actually give some sort of result and that is of course a lot harder than most papers will have you think. Until now I have just been finding my bearings in the lab (e.g. where do you keep the protease inhibitors? What? You freeze down your protein ladders/markers in the -80 °C freezer? Why can’t I find the antibodies in the boxes they’re meant to be in according to the Excel sheet? Etc.)

Luckily, people in the lab are all friendly and willing to help out. Despite that I still managed to crush a bottle in the centrifuge, by spinning it at 10,000 times the gravitational force. Turns out that it doesn’t need to be spun that quickly and quite evidently can’t take those forces. And this was already the second try. The first time I actually managed to rupture the bottle, losing some of my sample…

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Then of course there are the always unpredictable Western blots, as already alluded to earlier, in which one can investigate whether particular cells express certain proteins of interest. Ideally, this sort of experiment should yield a straightforward answer (more or less), as on the left of the following picture. But who even knows what happened on the right.

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Slotted between these attempts at lab work I’ve attended training to use the fancy flow cytometers, which can analyse single cells and even sort them according to different properties, and confocal microscopes. And so, let the next week of lab commence!

A bit more pancreas

Last time there was an introduction to the overall anatomy of the pancreas, its basic functions and some of its diseases. Today we will go into a bit more depth but still keep it relatively simple.

Firstly, what does it look like inside a pancreas when one zooms in a bit more? In the following diagram the exocrine cells are shown in red – these are divided into:

  • Acinar cells. They are found in clusters that sometimes resemble bunches of grapes. In the diagram the blue spots represent the nuclei (where DNA is kept). However, these are actually found on the side of the acinar cells that does not face into the duct (i.e. the basal side; the apical side faces into the duct/lumen).
  • Ductal cells. These line the ducts through which the digestive enzymes – produced by the acinar cells – pass.
  • Centroacinar cells. A small group of cells that reside at the boundary of an acinus and a duct.
Diagram of exocrine and endocrine parts of the pancreas - copied directly from Wikipedia here

Diagram of exocrine and endocrine parts of the pancreas – copied directly from Wikipedia here

The endocrine portion of the pancreas, on the other hand, is shown in turquoise. These cells are organised into islets and secrete the hormones they produce directly into the bloodstream. They consist of:

  • Alpha cells. Produce the hormone glucagon, which opposes insulin and raises blood glucose levels.
  • Beta cells. Produce the hormone insulin, which decreases blood glucose levels by stimulating the uptake of glucose into muscle and fat cells.
  • Delta cells. Produce the hormone somatostatin, which opposes a lot of other hormones in the body and, in particular, can slow down digestion.
  • Gamma cells. Produce so-called pancreatic polypeptide, which regulates secretion of other hormones in the pancreas.
  • Epsilon cells. Produce the hormone ghrelin, which is also known as the “hunger hormone” and signals to the brain that the stomach is empty.

Overall the two parts of the pancreas perform complementary roles: the exocrine pancreas helps to physically digest food coming from the stomach, whereas the endocrine pancreas makes sure that the glucose released into the blood due to the digestive processes is used/stored properly.

Secondly, people who are not scientists sometimes ask me how we actually study both normal and disease processes in the lab. Focussing on pancreatic ductal adenocarcinoma (PDAC), here is a brief overview of the systems used in the lab to gain a better understanding of what is going on in human disease.

  1. The easiest system to use are so-called (pancreatic cancer) cell lines – these are cells that were once taken from a patient and then cultured in the lab (in vitro). So although they are originally from a very relevant biological setting, the continuous culturing in the lab means that they are no longer physiological. For example, they grow in flat sheets on plastic trays, do not interact with any other cells and often have mutations that go above and beyond what is found in a real cancer. However, they allow relatively quick, controlled experiments to be conducted and are therefore still indispensable.
  2. More recently there has been a surge in research involving (pancreatic) organoids. These are mini-organs that are functionally closer to the real organ being studied and also have three dimensional structures that are more physiological. However, they need to be kept in relatively specialised conditions and are a little bit more tricky to manipulate than the cell lines.
  3. Since it is impossible (for a lot of reasons, including ethical ones) to conduct experiments on humans, cancer biologists most often turn to mice since they are also mammals and are genetically similar to us – we share approximately 90% of our DNA sequences. Mouse models of cancer allow complicated, intricate and biologically relevant experiments to be carried out, but they do take more time and need to be planned very carefully. Guerra & Barbacid (2013) have written a review specifically on PDAC mouse models.
  4. Data from human cancer biopsies. Increasingly, as sequencing whole genomes is becoming cheaper and easier to do, labs use DNA sequencing data from patients to compare some of their results from the mouse experiments. Biopsies can also be used to look at the histology (micro-anatomy) of certain stages or types of cancer in humans.

In the end, these techniques are used together, since all of them have strengths and weaknesses, allowing (hopefully) the most accurate description of what is going on in a certain (disease) scenario.

Lastly, what exactly do we use all these methods and systems for; what are the outstanding questions surrounding PDAC? Well, there are quite a few, but here are some of the more common ones: Why do most PDACs arise in the head and not the tail of the pancreas? Why are they so prone to metastasise (spread through the body)? How can we detect PDAC sooner and, importantly, distinguish it from inflammation of the pancreas? Where exactly (which cells) do PDACs come from? What roles do stem cells play in all of this? (If you have the answers to any of these questions, please do not hesitate to get in touch.)

Reference:

Guerra C, Barbacid M (2013) Genetically engineered mouse models of pancreatic adenocarcinoma. Mol Oncol 7: 232-247

An Introduction to the Pancreas and its Cancer(s)

Given that the four-year PhD I am about to embark on is being partly funded by Cancer Research UK (CRUK), why not start with a short video they have put together containing ten things about that pancreatic cancer that you may (not) have known:

Firstly, is there anything more to its anatomy than “being a small organ in the abdomen”? Why yes, funny you should ask. The pancreas reaches up to 15 cm in length in humans and is a J-shaped structure, apparently a bit like a hockey stick. The organ is not symmetric, rather it is made of a head (left), body (middle) and tail (right), as shown below. In the head the main pancreatic duct merges with the common bile duct before they join into the duodenum, where food is further digested after it leaves the stomach.

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Cartoon illustration of a human pancreas – copied directly from Hezel et al, 2006

As is often the case in biology – and this applies from the level of individual proteins to a whole organism – there is an intricate relationship between structure and function. The pancreas has two main functions: firstly, it contains endocrine cells that produce hormones, such as insulin, to regulate glucose balance (or homeostasis). Secondly, it contains exocrine cells, which make up the majority of the organ, that produce enzymes to aid food digestion. These two categories of cells therefore work together and coordinate the body’s metabolic response to food consumption.

Not all animals have pancreases as complex as those found in mammals since they have evolved over time. Worms, for instance, have some specialised gut cells that produce a substance similar to insulin. Ancient sharks and some fish contain organs in which the exo- and endocrine cells are found together. Fruit flies astonishingly produce insulin in their brains and, in particular, those cells develop from a completely different tissue in the embryo – called the ectoderm – than in other animals, where the pancreas stems from the endoderm. In amphibians and mammals the anatomy becomes more complex with characteristic “islets” of endocrine cells. (See Stanger & Hebrok, 2013 for more details.)

Since I am the daughter of two doctors I am usually interested in the pathological side of human biology. What sort of diseases befall the pancreas? Possibly the first that comes to mind is (type I, auto-immune type) diabetes in which the pancreas fails to make enough insulin because the body is attacking its own beta cells that normally produce insulin. Other than that the pancreas can of course become inflamed like any other organ and this is known as pancreatitis. It is caused, among other things, by high doses of alcohol. Lastly, the pancreas can become cancerous.

Although there are a few different types of pancreatic cancer the focus will lie on pancreatic ductal adenocarcinoma (PDAC) since it is both the most frequent and also the best understood type. It is one of the deadliest types of cancer because it often goes unnoticed until the tumour has disseminated (or metastasised) in the body – fewer than 10% of people who are diagnosed survive for longer than five years.

Therefore it is important to find out more about PDAC. But for the moment there is a lot we do know, decades of hard work that are often frustratingly glossed over. For instance, the genetic mutations that start and maintain PDACs are well known. Among them are activating mutations in a famous oncogene – a gene whose overactivation contributes to cell proliferation and cancer – called KRAS, and deletions of tumour suppressors – those genes/proteins that act as checks and balances and stop cells from overproliferating – such as p16 and p53. Moreover, it is well known when these mutations occur during the development of the cancer. The following image shows how some PDACs form: from healthy tissue to slightly abnormal tissue until it invades into the rest of the body.

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Histology of normal (left), dysplastic (middle) and malignant (right) pancreatic tissue – copied directly from Bardeesy & DePinho, 2002

As for treatment, the most effective method, if successful, is surgery. However, this often involves removing the head of the pancreas as well as parts of the duodenum and this, in turn, means that the stomach has to be surgically joined directly to subsequent parts of the digestive tract, making it a complex procedure. In addition, patients receive radio- and/or chemotherapy, which “targets” most proliferating cells. Targeted therapies that are specific to the cancer cells, as opposed to healthily dividing cells, are, as far as I am aware, not particularly established yet for pancreatic cancer.

For surgery to be effective the cancer needs to be detected at earlier stages. In other words, better diagnosis procedures need to be developed. Recently, two papers attracted some attention because they uncovered methods that could potentially make diagnoses easier using either blood (Melo et al, 2015) or urine (Radon et al, 2015) samples. However, as CRUK rightly point out, these techniques need to be refined for them to be able to tell true cancers from chronic pancreas infections.

And on that cheery note… Next time you can look forward to learning a bit more about how PDAC is studied in the lab and pancreatic anatomy in slightly more detail.

References:

Bardeesy N, DePinho RA (2002) Pancreatic cancer biology and genetics. Nat Rev Cancer 2: 897-909

Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, DePinho RA (2006) Genetics and biology of pancreatic ductal adenocarcinoma. Genes & Development 20: 1218-1249

Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523: 177-182

Radon TP, Massat NJ, Jones R, Alrawashdeh W, Dumartin L, Ennis D, Duffy SW, Kocher HM, Pereira SP, Guarner  L, Murta-Nascimento C, Real FX, Malats N, Neoptolemos J, Costello E, Greenhalf W, Lemoine NR, Crnogorac-Jurcevic T (2015) Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma. Clinical Cancer Research 21: 3512-3521

Stanger BZ, Hebrok M (2013) Control of Cell Identity in Pancreas Development and Regeneration. Gastroenterology 144: 1170-1179