Still digesting…

… the outcome of the Brexit referendum, yes. Or the fact that Austrian presidential elections need to be repeated, yes. But also, and on a more positive and scientific note, still digesting articles at eLife. It’s almost exactly a year since I did a short internship with their Features Editorial team, at the end of which my boss, Peter Rodgers, asked whether I would consider continue writing as a freelancer. Consider it? Of course. Yes, no consideration needed. I don’t think I could conceal quite how pleased I was with the offer. So for almost a year now I have been writing one digest a week (about two hours worth of work) and here I’d just like to highlight a few of the most interesting ones.

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation

This was the second paper that landed in my inbox to digest. When I read the subject line I was a bit baffled by the coincidence, because it surely had to be a coincidence. The lead author of this paper was none other than my current PhD supervisor with whom I was scheduled to start a month later.
The main finding of this paper was a little bit counter-intuitive. The first author, Sophia Blake, studied glioblastomas, the most aggressive form of brain cancer, iand found that when she deleted a tumour suppressor gene called p53 in mice, the animals developed these tumours. So far so good. However, when she deleted a second tumour suppressor called ATMIN at the same time, fewer mice got fewer and smaller tumours.  The paper then goes into some mechanistic detail of how this happens and finishes by showing that there are probably similar processes at play in human glioblastomas.

Ebola virus disease in the Democratic Republic of the Congo, 1976-2014

Most often the papers I read and digest are about cancer, stem cells or molecular biology. Here, however, I got to take a look at an epidemiology study: the authors compiled data for seven Ebola outbreaks in the Democratic Republic of the Congo. To me the most interesting observation was that outbreaks that had, at the outset, a high “reproduction number” – the number of people a single infected person transmits the disease to – were caught and contained early. However, when this reproduction number was smaller than about three the outbreaks seemed to be dealt with less quickly, leading to an overall greater negative effect.

Pericytes are progenitors for coronary artery smooth muscle

In this paper Volz et al. used fluorescent imaging to track the progression of epicardial cells (on the surface of the heart) deep into the muscle tissue of the heart. Using these microscopy techniques, the authors could follow how the epicardial cells become smooth muscle cells, cells that contract and relax, in the coronary arteries. Clicking on the image below will take you to a video consisting of snapshots taken from the outside of a mouse heart to further within. The epicardial cells first become so-called pericytes, cells that normally support blood vessels, and then eventually turn into smooth muscle cells.

pericytes

Snapshot from the first video in Volz et al.

Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

This last paper I want to mention briefly because it is on a subject that is similar to my project. Fessart et al. studied what can make lung and breast cancer cells more aggressive, more tumorigenic. They noticed that a protein called AGR2, which is normally found within cells where it helps to fold other proteins correctly, can also be secreted outside cells. When this happens AGR2 can make healthy lung cells cancerous.

Almost one year of PhD is already over, three more to go. I think we can count ourselves lucky if, by the end of it, we have a nice story to publish…

References:

Blake SM, Stricker SH, Halavach H, Poetsch AR, Cresswell G, Kelly G, Kanu N, Marino S, Luscombe NM, Pollard SM, Behrens A (2016) Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation. eLife 5: e08711

Fessart D, Domblides C, Avril T, Eriksson LA, Begueret H, Pineau R, Malrieux C, Dugot-Senant N, Lucchesi C, Chevet E, Delom F (2016) Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. eLife 5: e13887

Rosello A, Mossoko M, Flasche S, Van Hoek AJ, Mbala P, Camacho A, Funk S, Kucharski A, Ilunga BK, Edmunds WJ, Piot P, Baguelin M, Muyembe Tamfum J-J (2015) Ebola virus disease in the Democratic Republic of the Congo, 1976-2014. eLife 4: e09015

Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, Kofler N, Kitajewski J, Weissman I, Red-Horse K (2015) Pericytes are progenitors for coronary artery smooth muscle. eLife 4: e10036

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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.

Screen Shot 2015-09-04 at 22.08.59

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.

Screen Shot 2015-09-04 at 22.42.00

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

Organoids.

…finding the middle ground between using a whole genetically modified organism and a flat sheet of cells in a Petri dish…

According to Austin Smith, director of the Stem Cell Institute at the University of Cambridge and also known to his students as the stem cell guy, organoids are “probably the most significant development in the stem-cell field in the last five or six years”.

So what exactly are these futuristic sounding things? In essence, organoids are miniature versions of whole organs that can be grown in vitro, that is, in the lab/outside an organism. Growing these organoids works relatively well and easily for some organ types such as the gut/colon, the liver and the stomach. The main distinguishing feature between an organoid and a real organ is size – organoids rarely grow beyond half a millimetre in diameter. Partly this is because a vascular system, supplying oxygen and nutrients, would need to be integrated to allow larger organoids to grow. However, their three-dimensional organisation and functionality can be surprisingly similar to that of their real counterparts.

OK, great. But where do organoids come from? Although Aristotle proposed that living things could arise from inanimate matter (the theory of spontaneous generation) this way of thinking is generally accepted to have been disproven by Louis Pasteur and others in the nineteenth century. Since organoids do not just pop into existence in scientists’ Petri dishes, one has to grow them from something. Several different starting materials can be used: most easily or intuitively, organoids can be derived from tissue biopsies of the corresponding organ. Alternatively, one can extract adult stem cells from a (mouse) organ and let those cells grow in vitro using special cell/tissue culture conditions. Lastly, it is possible to “persuade” embryonic stem cells or induced pluripotent stem cells to become organoids, provided that one can supply the right signals in the growth medium.

Organoids – so what are they good for? One of the earlier studies (Sato et al., 2009) showed that it is possible to generate intestinal organoids from single intestinal stem cells, in effect backing up the researchers’ claim that there is a well-defined (so-called Lgr5+) stem cell in the gut. Firstly, therefore, organoids help answer questions in the fields of developmental and stem cell biology. A sample “mini-gut” of Sato et al. is shown below: it exhibits crypt-like structures (where stem cells reside and replenish the tissue), villus-like structures and finally, the spheres partly fold in upon themselves to mimic the lumen or interior of the intestine where food would be digested.

A mini-gut - copied directly from Sato et al., 2009

A mini-gut – copied directly from Sato et al., 2009

For the intestine, but also other organs such as the brain, stomach and pancreas, organoids can shed light on questions of basic anatomy: organs are highly complex and often difficult to study in vivo/inside an organism and for some organs we still lack surprising amounts of knowledge. For example, McCracken et al. (2014) used human pluripotent stem cells to grow an organoid that resembles the first half (antrum) of the stomach. Next it will be interesting to find out how to create the second half (fundus), and this, in turn, will help explain how a whole stomach and, eventually, gastrointestinal tract develops.

However, organoids are also being used to study diseases such as cancer, diabetes and neurological disorders. For example, Boj et al. (2015) took biopsies from both mouse and human pancreases (or pancreata, if you are feeling particularly confident) and could obtain corresponding pancreatic organoids. In particular, these organoids recapitulated several different stages of pancreatic cancer and could be genetically modified as well as used to discover new ways in which cancer cells are abnormal.

How to make a pancreatic organoid - copied from Boj et al. (2015)

How to make a pancreatic organoid – copied directly from Boj et al., 2015

In particular, the hope is that, if making organoids becomes fast, cheap and easy, it can be integrated into medical procedures. Van de Wetering et al. (2015) have attempted this for colorectal cancer. In their paper they describe the creation of a “living biobank” of cancerous intestinal organoids from twenty cancer patients. Since the organoids come from a specific patient’s body it can be used for in-depth analysis of mutations, but also for drug testing to see which of the available drugs might work best for the patient in question. This approach is known as personalised medicine and despite being talked about a lot by scientists and the media alike, has yet to really take hold in hospitals.

This crucial last step – from “bench-to-bedside” – really seems to be the major obstacle stopping cool discoveries in the lab from becoming useful in hospitals/treatment settings. Training more medical doctor/PhDs (MD/PhDs), which is far more common in the US than in the UK, would certainly help. These people are able to appreciate the problems (or challenges, if you like) as well as the needs on both sides. But even then, growing organoids for each and every cancer patient would be a huge logistical undertaking, so there would have to be a lot of investment as well. Maybe initiatives such as these “organs-on-chips” – testing drugs on organoids in an organised, high-throughput manner – will bridge the gap?

References:

Boj Sylvia F, Hwang C-I, Baker Lindsey A, Chio Iok In C, Engle Dannielle D, Corbo V, Jager M, Ponz-Sarvise M, Tiriac H, Spector Mona S, Gracanin A, Oni T, Yu Kenneth H, van Boxtel R, Huch M, Rivera Keith D, Wilson John P, Feigin Michael E, Öhlund D, Handly-Santana A, Ardito-Abraham Christine M, Ludwig M, et al. (2015) Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell 160: 324-338

McCracken KW, Cata EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai Y-H, Mayhew CN, Spence JR, Zavros Y, Wells JM (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516: 400-404

Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459: 262-265

van de Wetering M, Francies Hayley E, Francis Joshua M, Bounova G, Iorio F, Pronk A, van Houdt W, van Gorp J, Taylor-Weiner A, Kester L, McLaren-Douglas A, Blokker J, Jaksani S, Bartfeld S, Volckman R, van Sluis P, Li Vivian SW, Seepo S, Sekhar Pedamallu C, Cibulskis K et al (2015) Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients. Cell 161: 933-945

PhD Interview for the Francis Crick Institute!

Despite being funded as a Cancer Research UK charity, the London Research Institute (LRI) went to considerable lengths to ensure that we interviewees were comfortable during our three-day visit to London and the institute. Firstly, our travel expenses – ranging from short intra-England train journeys to flights from across Europe and North America – were covered, as well as our accommodation at a hotel overlooking Russell Square at the heart of Bloomsbury:

image1

The first day was probably the most strenuous. First we listened to introductory talks given by the LRI Academic Director and the LRI’s Deputy Director who, incidentally, also quoted Donald Rumsfeld about the unknown unknowns just like at the departmental research day. Furthermore, as part of my destressing strategy I took a walk around the area during one of the breaks, inevitably stumbled into a bookshop and found this:

image2

The rest of the first day was filled by talks given by each of the recruiting group leaders. Eighteen times ten minutes of concentration. After that we got the chance to speak to those principal investigators (PIs) we were interested in. Lastly, we had dinner with the PIs and some of their students. And although all of this was not part of the “official” assessment procedure I think it was important to be making a good impression throughout, and therefore by the end of this first day most of us felt exhausted.

The official panel interviews were scheduled for the second day. We each had to give a presentation of a research project we were involved in, as well as a critique of a research paper. We were then asked some questions on these presentations and also had the usual questions hurtled at us, “Why do you want to do a PhD? Why do you want to do it at the LRI? What are your long-term goals?” Etc.

We were also privy to a tour of the LRI building at Lincoln’s Inn Fields. During the introductory talks they emphasised how great the facilities – DNA sequencing, flow cytometry and microscopy among others – at the LRI are. I was skeptical at first, but the tour was convincing, especially considering that probably not so much money is being invested in the upkeep of this building due to the move of the LRI into the Francis Crick Institute in 2016. At the Crick of course everything will be even better, as they didn’t fail to mention at every possible opportunity.

On the second day we had dinner together with the lab members of the recruiting labs, but without the PIs who were busy trying to work out who to invite for the third day on which one-on-one interviews would be held. We were certainly more relaxed this evening. However, the next morning between 7.15 and 8.00 am we had to come down into the reception area of the hotel to pick up a letter informing us whether we had been invited for the third and final day. It was irrational to be nervous because at this stage there was absolutely nothing to be done about the situation. Nevertheless, I, and probably many others, had difficulty sleeping that night.

Luckily, I was invited back to speak to three group leaders: Axel Behrens, Victoria Sanz-Moreno with Ilaria Malanchi, and Caroline Hill. In these sessions it became clear that I would want to work either with Axel on pancreatic cancer or with Victoria and Ilaria on melanoma. The third project was more focussed on neurodevelopment, which is interesting but my gut feeling told me to veer away from it simply because I have a stronger background in cancer biology.

At the end of the third day we had to hand in a preference list, and then all there was left to do was to go back to Cambridge and wait. But the waiting was mainly a formality since it had become clear during the day that Axel Behrens’ lab was going to make me an offer I couldn’t refuse. I am extremely excited! London, here I come!