This is going to be a long and technical one sorry.
This month I want to talk about an experiment I've done pretty much every day for the last three months (plus continuously over the last 2.5 years) - Western Blot.
Western blots are used to visualise and identify individual proteins. In this technique you have a solution (called a lysate) filled with the majority of proteins from cells. This technique separates these proteins out by size and allows you to stain for specific proteins you want (more on this later). This is the standard way to visualise proteins but it is very subjective and is tricky to perfect (I hope you will appreciate why after reading this).
The proteins are visualised by staining them with antibodies specific to that protein and then using a chemical reaction to produce light which can be picked up by a machine. The resulting image is of black bands. The intensity of the black band determines the approximate level of protein present.
There are a few steps to Western Blotting which I will go through. The whole technique can take 1-3 days depending on what proteins you are looking for and what you choose to do in each step.
Steps to Western Blotting:
A little bit of terminology before we begin:
STEP 1: Preparation
Reagents and Buffers
Before you begin a western you need to make sure you have all your reagents prepared. I will NOT be going through this, all I will say is you need reagents for
When you extract your protein from your cells you end up with a protein lysate. This contains the majority of the proteins in your cells. You don't know what the concentration of the total protein is but you are sure that each sample doesn't contain the same concentration. This is due to how well the extraction went in each individual sample but also how many cells there is to extract protein from. There are a few ways of finding out the concentration of your protein which I won't go through (I use the Bradford Assay from BioRad).
What you do need to know is that for each sample you need to make up a solution of protein, water and loading buffer to load onto your gel. You can use as much or as little protein as you want, I typically use 10-15ug per well. I load 20ul per well so I need:
There are many types of loading buffer, I use Laemmli buffer.
The loading buffer has a couple of functions. You are using an electric current to make the proteins travel through the gel. Proteins are all different charges so the buffer makes sure all the proteins have the same charge (negative) - this is done by a chemical called SDS. Proteins are globular structures. These don't run though a gel very well so you need to unravel the proteins into single strands (called denaturation). The buffer will start this process - a chemical called β-mercaptoethanol breaks disulphide bonds. The loading buffer also has a dye in it. This is called the "loading front" which runs faster than your proteins. This allows you to track how far your proteins have travelled through the gel (image later).
A quick note - usually the protein samples you have prepared will be blue once you add the loading buffer and boil it. This is because the dye in the loading buffer is blue. However, the protein I will show in this post will be yellow. This is because I extracted the proteins using acid and the acid in the lysate turns the buffer yellow. Once the gel starts running the dye front goes back to blue.
Gels are made up of:
The acrylamide will set into a gel which has pores in it. The protein will travel through these pores. The percentage of acrylamide used determines how big these pores are. Large proteins run slowly through a gel whereas smaller proteins will run very quickly through the gel. A lower percentage gel (e.g. 7%) will have large pores, allowing larger proteins to run faster through the gel. However you are likely to lose all the smallest proteins. A higher percentage gel (e.g. 12.5%) will have very small pores which slow down the small proteins, allowing you to visualise them easier. However all your largest proteins might not run as they can't past through the pores.
There are two types of gels used in gel electrophoresis - a stacking gel and a resolving gel. They're pretty much made up of the same ingredients (a stacking gel is slightly more acidic pH 6.8).
Steps to making a gel:
I have made up a 12.5% gel for this example to run histone proteins which are approx 15-17kDa in size. I will also stain for a loading control, beta actin, which is 45kDa in size.
STEP 2: Loading and running SDS-PAGE Gel Electrophoresis
The next step is to load your prepared protein into the wells in your stacking gel and running an electric current through the gel to allow the proteins to travel from negative to positive.
The gel(s) are clamped into an electrode chamber and placed in a running tank. The centre is filled with running buffer. The running buffer allows an efficient flow of electrical current to pass through the gels. It also prevents the gels from drying out which you do not want.
STEP 3: Transferring proteins from gel to PVDF membrane
Once the gel has run enough you can stop it. This depends on how long you want to wait for and what proteins you're looking for. My proteins are very small (17kDa) so I can stop my gel once I see the 17kDa ladder band is far enough from the loading front.
The next step is to transfer the proteins from the gel onto a membrane. You cannot stain for your proteins on the gel as it is far to delicate and not permeable - the protein only runs through the gel because we created the wells. The membrane is more permeable and allows you to stain for proteins.
There are a couple of (messy and time consuming) ways to transfer proteins but I prefer to use the iBlot dry technique. This basically transfers the proteins using a machine in 5 minutes (compared to 1-2 hours). The transfer stack consists of:
Again an electric current is used to push the proteins through the gel into the membrane. However the voltage is very low. As we know, small proteins move very quickly. The low voltage prevents the small proteins from passing straight through the membrane.
So how does it work:
After the transfer is complete you need to be VERY fast. You have to quickly remove the stack from the machine, get the membrane out of the stack and place it in TBST. The membrane CANNOT dry out or else all is lost. You can check your transfer at this stage using Ponceau red, which is a red dye that shows up all the wells and bands of proteins but I did not do that today.
STEP 4: Blocking
The next step is to block any unspecific binding. The antibodies are usually polyclonal - meaning they can bind to other proteins that aren't your protein of interest. To prevent this and also to get a nice clean image at the end, you block unspecific binding.
You can block using either of these two reagents:
The reagent you are using depends on the antibodies and proteins you are looking for. I block in 5% BSA/TBST/NaN3.
Before I block I cut my membrane so that I have the two individual gels again. You don't have to do this but I like to. At this point I just cut the excess off using the ladders as a guide. I then mark with a pen on the ladder what gel it is.
The membranes are blocked for 1 hour at room temperature on a rocker.
This is now when we can call out membrane a blot.
After blocking you can cut your blots to stain for multiple proteins. In this case I cut at 36kDa band (blue band above the bottom red band on the ladder) to stain for my histone proteins and also my beta actin loading control.
STEP 5: Antibody Staining of Proteins
This technique as I said uses antibodies which bind to specific proteins and you can them visualise these antibodies using a chemical reaction.
The figure below gives a rough idea of how the technique works.
The antibodies are created in different animal models (please don't ask how cause I have no idea). So if your primary antibody is developed in a rabbit then the secondary antibody will be goat anti-rabbit (i.e an antibody developed in a goat to bind specifically to rabbit-developed antibodies). There are some human antibodies but they're incredibly expensive.
The primary antibodies are made up in the buffer recommended by the manufacturer usually 5% BSA/TBST/NaN3. The recommended concentration is usually given by the manufacturer - typically 1:1000 or 1:2000 (i.e. 1ul antibody in 1ml solvent).
The secondary antibodies are made up in usually either 5% BSA/TBST or 5% Milk/TBST. The BSA doesn't contain NaN3 in the secondary because sodium azide (NaN3) inhibits the secondary antibody. The concentration of the secondary depends on how abundant the protein is and how long you develop it for. For example beta actin, which makes up part of the cytoskeleton of your cells, is an incredibly abundant protein so the secondary concentration is 1:50,000 BUT another protein I look at p-AMPK is not so abundant and the secondary concentration could be as low as 1:1000.
Part 1: Primary antibody incubation and washing
NOTE: for beta actin, you do a 1 hour incubation at room temperature
Part 2: Secondary antibody incubation and washing
For the secondary antibody you can do this at room temperature for 30 minutes - 1 hour, again on a roller.
I make up most of my secondaries in 5% Milk/TBST, except p-AMPK. The casein in the milk removes phosphorylation from proteins so I would lose my p from the p-AMPK if I did the secondary in milk. So for p-AMPK and all phosphorylation proteins I use 5% BSA/TBST.
Again after incubation in the secondary, the blot(s) need to be washed at least 6x5 minutes to remove excess secondary antibody.
The washing part of the procedure is honestly the most time consuming and tedious. It's a lot of back and forth to and from my office desk to the rocker. I usually try to do something else in the lab to not waste the time but sometimes it can't be avoided.
STEP 6: Developing
The final step (thank god) is to develop your blot. This step adds the peroxide and luminol enhancer solutions (collectively known as ECL solutions) to the membranes. You then take the membranes to a chemiluminescence machine which will detect the light emitted and give you an image in return.
You end up with a picture which has black bands. As I said at the start the intensity of the bands indicates how much protein is present - very dark means a lot, very light means very little and nothing means nothing.
The image above shows the loading control (beta actin) for a blot I have done for two cell lines, MCF10A (lane 1-3) and MCF12A (lane 4-6). The bands are very dark indicating that the protein is very abundant. What's important is the bands are almost the exact same size and intensity as each other (different in the two cell lines) indicating that my loading was very even (wahey).
Once the blot is developed you can keep it or you can throw it away. If you keep it and want to stain for another protein or the same protein but with a different secondary concentration you need to strip the blot (i.e. remove all antibodies bound to proteins), re-block using the same blocking buffer and re-incubate with the primary and secondary antibodies. A blot should only really be stripped twice. This will add another day onto your experiment (at least).
And that is it. That is roughly how a western blot works.
The reason why western blots are THE most hated laboratory experiment is because they rarely go the way you want them to, there is always a difference each time you do it and it takes FOREVER.
If a western goes wrong most of the time you have no idea why. It can be anything from one specific buffer which was made up wrong to the wrong gel percentage, the wrong transfer time or transfer voltage etc. Basically everything you did could have an issue associated with it. Trust me I did troubleshooting for TWO MONTHS on one antibody. It eats away at your soul.
Western blotting everyone.
Happy New Year!!
January is the month where New Year's resolutions are made...and broken. We all want to get fit, eat healthier and improve our wellbeing. A brilliant campaign that combines fundraising, awareness and improving your health is Dry January. Many charities encourage people to stop drinking alcohol for one month in order to raise awareness about a number of different diseases linked to alcohol consumption.
Since I am a cancer researcher (in case you haven't noticed) I am going to talk to you about how alcohol can increase your risk of cancer and why moderation is always key!
What is alcohol?
What do I mean by alcohol increasing risk and does this change depending on the type of drink? Are light beers better for you compared to vodka?
So what is alcohol? Well interestingly alcohols are not the beverages we think of but a group of chemicals all sharing similar properties (they all contain CH3OH). So methanol, ethanol and propanol are all members of the same alcohol family. When we talk about the beverage "alcohol" we are talking about ethanol. Now 100% ethanol is extremely toxic and flammable. You should NEVER drink 100% ethanol, the majority of alcoholic beverages range between 2% - 40%. For reference, I use 70% ethanol to clean my TC hood to remove cells and microbes to maintain the sterility of TC. Actually in the lab we use ethanol for a number of things including "washing" DNA. The way we extract DNA means there are chemicals mixed up with the DNA (like isopropanol). We want our DNA to be as pure as possible so to get rid of those chemicals we "wash" the DNA (in the form of a pellet) with 70-80% ethanol and allow the residual ethanol to evaporate off, leaving us some (hopefully) pure DNA.
Alcohol increases cancer risk and incidence of cancer:
Alcohol has been shown to increase the risk and actually cause seven different cancer types (shown below), but has been implicated in more cancer types. In fact when we talk about the major risk factors for any cancers the top three is always age, BMI and alcohol consumption.
The impact of alcohol on cancer depends on the cancer type, for example studies have shown that if a woman drinks 1.5 units a day her risk of breast cancer increases by 5%. Now that doesn't seem like a lot but that percentage increases the more your drink. The risk for cancers such as liver or mouth cancer have a far higher increased risk of cancer with just 1.5 units a day (liver cancer is 16%).
How does Alcohol cause Cancer?
Alcohol is classified as a carcinogen (since 1988 by IARC). Carcinogens are substances that cause damage to any part of a living cell. Alcohol is metabolised in the cell to acetaldehyde and this has the carcinogenic properties. There are a number of ways alcohol can cause cancer:
As I've talked about before, cancer in general is an accumulation of small changes to normal biological pathways such as DNA damage, changes in hormone levels, changes in nutrient levels and over activation of certain pathways. This all contributes to the cells becoming cancerous. Alcohol can generate and help increase these pathways, making it easier for cells to become cancerous. In fact alcohol actually increase risk further when combined with smoking (something people commonly do when they drink). This is because cigarettes contain other carcinogens which also damage DNA etc. So you're getting a double hit of damage at once.
What is the recommended intake of alcohol?
We hear a lot about the "recommended units of alcohol" per person, per week. But what is that?
Well first of all a "unit" is a measurement which depends on percentage alcohol and volume of the liquid. So the unit depends on the type of alcohol you're drinking, for example 1 unit of spirits is 25ml volume of 40% alcohol but 1 unit of beer is about 225ml of 4% alcohol.
The new guidelines (released a couple of years ago) recommend that men and women should drink no more than 14 units of alcohol per week BUT stipulates this must be spread over at least THREE DAYS. Meaning for example you can have 6 glasses of wine a week but you can only have four glasses max in one day. This is to try reduce binge drinking. Binge drinking is defined by the Office of National Statistics as over 8 units of alcohol for men in a single sitting (about 3 pints) or 6 units for women in a single sitting (about 2.5 glasses of wine) or drinking a large amount of alcohol in a short period of time.
The take home message:
It's all about moderation! If you enjoy a glass of wine in the evening that's fine but maybe one day of the week you don't have that glass of wine! It's important to be aware of the volume you are drinking. Pints are easier because pint glasses are standardised but wine glasses can come in different shapes and sizes. How do you know you're drinking 175ml?
It's also about feeling better and improving your overall health. Alcohol is not just linked to cancer but other diseases like cardiovascular disease. Alcohol can also contribute to weight gain, especially high sugar drinks like larger, cider and/or wine. Ask anyone who has done Dry January before and they will tell you how good they feel, even after just a few weeks.
I'm not preaching here. I'm not saying GIVE UP ALCOHOL NOW! I enjoy a drink but I don't drink very often as a choice.
What I am suggesting that maybe reducing you alcohol intake could improve your health and overall lifestyle for the better. January is the month to try it out while you're surrounded by other "designated drivers", see how it benefits your health (and your pocket).
If you want to become a Dryathlete or just find out more about the campaigns you can visit:
November is a month of awareness for a few cancers. I will discuss each briefly but you can find more detail in the links given below.
I'm going to start with the worst here, pancreatic cancer.
Pancreatic cancer is the 11th most common cancer in the UK accounting for about 10,000 cases each year. The incidence has been steadily increasing over the last 10 years, more so in women than in men. It only accounts for about 5% of cancer-related deaths each year but here's the kicker...less than 1% of pancreatic patients will survive 10 years or more. The five-year survival rate? 3%. And finally only 20% of patients will survive their first year. That is a loss on average of around 8,000 lives a year just down to one cancer type.
So why is this cancer type so bad. There are a number of reasons, the first being your pancreas is pretty important for your survival. Your pancreas does two vital things, it produces enzymes which help you digest your food and it produces hormones to regulate your blood glucose levels. Hormones like insulin. The second reason pancreatic cancer is so hard to treat is its location. Your pancreas is embedded deep in your abdomen between your stomach and your spine. The third reason is your pancreas doesn't regenerate and it's very difficult to replace with a donor. And finally and possibly most importantly for cancer treatment, pancreatic cancer usually presents at a very late stage meaning very little can be done.
Again with a lot of cancers the symptoms are vague and can include:
Type of pancreatic cancer:
Diagnosis and treatment:
Diagnosis of pancreatic cancer is mainly through examination of the pancreas using an ultrasound or CT scan accompanied by blood tests and a biopsy. The majority of patients are diagnosed at stage IV, which is why the survival rate is so low. The treatments for pancreatic cancer are the standard: surgery, chemotherapy and/or radiotherapy. Unfortunately lack of response to treatment and recurrence is high. There are many ongoing clinical trails to find new targeted therapies for pancreatic cancer.
Screening and prevention:
Currently there are no screening programmes for pancreatic cancer and early diagnostic tools like CA19-9 are unreliable as a proportion of pancreatic cancers don't release this marker.
This is a terrible disease with a shocking survival rate and it is only getting worse. Pancreatic cancer research is one of the most underfunded research areas today. But with more awareness hopefully charities like Pancreatic Cancer UK can raise more funds for vital research.
To keep with the theme of vastly underfunded research areas I will next talk about
You would expect through all the campaigns about quitting smoking etc. that lung cancer would be one of the most funded cancer research areas but it is sadly not.
Lung cancer is the third most commonly diagnosed cancer in the UK accounting for about 46,000 new cases a year. But just like pancreatic cancer, the 10-year survival rate is only 5%. The sad thing is CRUK estimates about 89% of lung cancer cases could be prevented. I won't lie to you we all know the biggest risk factor for lung cancer is smoking. We've seen the ads and the photos on cigarette packets. The statistic here to remember is that while only 10% of smokers will get lung cancer a whopping 86% of lung cancer patients are smokers, former smokers or passive smokers.
Symptoms of Lung Cancer:
Types of lung cancer:
The "types" of lung cancer are grouped together because of the size of cell the cancer originated in but they are not all the same cancer.
Diagnosis and treatment:
Usually once you have presented with symptoms which indicate lung cancer a patient will receive a chest x-ray and/or bronchoscopy accompanied by a biopsy. It must be noted that treatment for the two cancer types are different, depending on where it is and the stage. But the standard treatments for lung cancer is standard: surgery (which could be as little as removing the tumour to as much as removing a lung), chemotherapy, radiotherapy, photodynamic therapy and ablation. There are targeted therapies available for advanced lung cancer patients which target pathways such as EGFR (erlotinib) or tyrosine kinases (genfitinib).
Screening and prevention:
While there is no national screening taking place for lung cancer the best way to prevent it is to quit smoking. Anti-smoking campaigns are one of the most successful awareness campaigns out there.
And the big question you've been burning to ask - why is lung cancer so underfunded? Sadly I have no answer for you. The funding process is a difficult and complex system which I know very little about. The good news is that CRUK at least have pledged to put more funding into these poorly researched cancers like Lung and Pancreatic.
And finally let's talk about Movember. Now November isn't the month for awareness on testicular cancer or prostate cancer so I won't be talking about these. However it has become a month where awareness and funds are raised for all men's cancers. And awareness to me is more important that a single penny. I have spoken on this before but just to hit it home once again, prevention is better than cure. Not all cancers can be prevented I'm afraid but if we can reduce the numbers of people being diagnosed we can reduce the numbers who lose their lives.
The fact the Movember has come about is particularly important. The most funded cancer research area to date is breast cancer. And it is thought that part of the reason for this is women. Women who had/have breast cancer or women who have lost a family member or friend to breast cancer. These women are the driving force behind every awareness campaign, behind new drugs being made available to patients and behind vastly successful national screening. To me it seems women are, interestingly, the major focus of cancer awareness campaigns because they are usually the driving force behind them. I'm not going to go into gender biases about how "women talk more about their problems than men" because if cancer screening has taught me anything its not true (take a look at bowel cancer screening statistics). But what I am saying is that men as a whole (sorry to put it like that) have less opportunities to avail of screening services and may not know what they are really supposed to be looking out for. Which is why Movember is so brilliant. It encourages men to get involved and to talk about male cancers. It puts male-specific cancers in focus while allowing men to actively participate by growing beards (super manly).
I like to highlight charities and companies that inspire me and to me show genius and innovative ways of raising cancer awareness and funds. And the underwear company OddBalls encompasses this completely. The company, started in 2014, designs and creates underwear (not just for men but for women as well). And with your new boxers you also receive a handy guide to checking yourself for testicular cancer. They have become so popular they now design underwear for rugby clubs including The Welsh International Team AND have set up their own charitable foundation.
If you want to know more or maybe by a pair for yourself, check out: www.myoddballs.com/
They also do socks, bobble hats, rugby jerseys and more!
If you have any questions please don't hesitate to contact me.
For more information visit:
October is breast cancer awareness month. I want to tell you in this post some information about breast cancer symptoms, diagnosis and treatments. I have worked in various areas of breast cancer research in the last 5 years, most notably my PhD is focused on understanding diabetic drugs in order to prevent breast cancer. It's an area close to my heart.
Breast cancer is the most commonly diagnosed cancer in the UK. About 55,000 new cases are diagnosed every year and 7% of all cancer-related deaths can be attributed to breast cancer. There is some good news and bad news about breast cancer survival. The overall 5-year survival is around 86% but when you break this down by stage of the cancer type, this value falls dramatically for those with higher stage cancers. 5-year survival for women with stage IV cancer is approximately 15%. The survival rate has doubled in the last 40 years due to better preventative measures, better diagnosis and better therapies.
Breast cancer types:
I've already spoken a bit on the different cancer types and how tumours can be classed by their origin but also molecular differences ("What the Hell? - Cancer Part 2).
I've also talked before about risk factors, which are lifestyle or genetic factors that can increase or decrease the likelihood of developing a certain disease. There are a number of risk factors associated with breast cancer (some are in image 3).
Most women know the symptoms of breast cancer:
There are a number of ways a breast tumour is identified:
Treatment depends on the stage/grade of your tumour, the molecular subtype and the willingness of the patient. There are the standard treatment practices (1-3) but also more personalised therapies (4-5)
Prevention is key. You will hear my harp on about prevention is the cure to cancer but really it's one of the key areas we as individuals can look after for our own health. In fact it’s estimated that about 27% of all breast cancers could be prevented and there are a number of prevention techniques you can adopt
All rights to this video belong to Coppafeel!.org
And let's not forget the men here! Men can suffer with breast cancer. While only about 400 men are diagnosed each year that is still a significant number. Male breast cancer is quite similar to female breast cancer in terms of risks (though an additional risk factor is Klinefelter's syndrome), diagnosis, symptoms and treatments.
Finally I would like to talk to you about a charity which inspires me to keep doing the work I do. This charity was set up by a young woman who was diagnosed with breast cancer in her early 20's. Instead of wallowing in her diagnosis of metastatic breast cancer she decided to set up a charity to better educate young women to CHECK YOUR BREASTS! That charity was set up over 8 years ago and despite multiple metastatic tumours Kris is still alive and more vibrant than ever. This charity is called Coppafeel! (a genius name). If you have time please check out their website https://coppafeel.org/ and check out Kris's story (https://coppafeel.org/our-charity/kris-story/). A truly inspirational woman backed by the most incredible fundraisers and supporters. I haven't had the chance to work with coppafeel but it is one of the charities I would most like to get involved in.
For more information on any of the areas I've discussed visit:
The staff and the organisers of the event were brilliant and so accommodating. They have this wonderful sort of art installation where the public/researchers at the Crick ask "What...the one thing [they] would like to know about the human body, how disease develops or how they might be treated?" I found one I thought was very insightful from a 17 year old, "Do atoms have motives?".
The first day opened with two key note speeches from two extraordinary researchers.
The first speaker, Dr Lewis Cantley, discovered the PI3K signalling pathway (which is a big deal trust me, this is one of the biggest signalling pathways there is and it is incredibly important in cancer biology). He actually spoke about something very close to my own research. Insulin resistance (hyperinsulinemia) is a risk factor for many types of cancers. The liver, skeletal muscle etc. become resistant to insulin signalling, leading to a build up of insulin in the blood. BUT cancer cells are highly sensitive to insulin, meaning this high blood insulin environment is perfect for the cancer to grow in. Insulin itself activates pathways to make cells grow and divide. PI3K is part of the insulin signalling pathway. Dr Cantley talked about how using PI3K inhibitors (which are already being used but can become ineffective) in combination with a modified diet could help enhance PI3K inhibition for cancer treatment. And funnily enough this PI3K and metabolism subject came up a lot throughout the two and a half days, not just in the "Metabolism" session.
The second speaker was Dr. Lisa Coussens, who is a big name in cancer immune biology. Dr. Coussens spoke about the immune cells in the tumour microenvironment and how this can contribute to inflammation around tumour cells. She also spoke about how immune cell infiltration into tissue can contribute to tumour development and metastasis. Understanding the different immune cell infiltrates will help is understanding tumour development and also potential treatment.
The second day was long, very long (11 hours from arriving to leaving). The day was packed with talks and poster presentations. I won't go through every single speaker's work in detail but I will highlight some areas I found particularly interesting. My main reason for attending this conference was the session on metabolism. I have five pages of small scribbled notes in my notebook from the five talks compared to one or two for the other sessions. Not the other sessions weren't interesting. Unfortunately I can't really discuss a huge amount of what was talked about because most of the data is unpublished and you have a professional courtesy not to disclose the information.
Session 1: Metabolsim
This session really focused on how understanding metabolic processes, the metabolites that are used in these processes and the metabolites derived from those processes could help in not only understanding how tumours are formed but also how was can target these tumours as individual treatments themselves or in fact enhance current treatments. As I've said before it's been shown that different diets can enhance the effect of anti-cancer drugs.
Session 2: Tumour Microenvironment
I've talked about the tumour microenvironment (TME) before in my "What the hell? - Cancer Part 1" post. The TME is made up of a number of different cell types (immune cells, blood vessel cells etc.) which all contribute to the development, growth and spread of cancer. In this session we discussed how we can understand the interaction between the cells in the TME and cancer cells. Mainly this session focused on infiltrating immune cells (immunology is a hot topic now so it came up in pretty much every talk at some point....) but there was one talk which was very interesting about identifying mutational signatures in breast cancer that can identify the type of cancer it is, can put together cancers that may not look the same but have similar mutations and can tell you about potential treatment pathways (Serena Nik-Zainal).
Session 3: Tumour Immune system interactions
As I said immunology in cancer is a hot topic in recent years. This session focused on targeting immune cells to kill cancer. Immune cells can be pro tumour and anti-tumour depending on the signals it received. Cancer cells can send out signals that activate immune cells that dampen down the immune response while also pretending they're normal cells to immune cells that would kill them. Immune cell infiltration into the tumour can happen at different stages of tumour development and the context (I learnt) is pretty important for choosing what cells to inhibit to kill cancer cells. Interestingly the metabolism/immunology sessions combined for a talk by Luke O'Neill (a lecturer in Trinity College Dublin). While by far the most entertaining lecture he also made an interesting point about how different metabolites can be pro-inflammation or anti-inflammation. While he doesn't focus on cancer, he made the interesting point that targeting immune cells could be again achieved or enhanced by looking at the metabolism of the cell you're targeting.
Session 4: Tumour heterogeneity and evolution
The final session was a mixture in terms of biology and statistics. We had talks about cancer evolution, specifically the evolution of drug resistance (drug resistance is a big problem in all cancers) and heterogeneity of cancers. If we can understand how drug resistance occurs we could target cancer cells better so it doesn't happen. A lot of that has to with the fact that tumours can have multiple cancer cell sub-groups which have different mutations (heterogeneity). The statistics talk was interesting but I was very much out of my depth. What really fascinated me and kind of scared me was the talk on brain tumours. Basically brain tumours act like mini-brains. They form cell-to-cell connections through micro-tubules. This tubes allow the cell mass to become more resilient to therapy (e.g. radiation) and also allow repair of damaged parts of the cell network (which brains do not do). Frank Winkler showed videos of the brain tumour cells and even compared them to normal brain cell connections. While scary to know, this is brilliant for brain tumour biology and treatment. Recurrence is common in brain tumours and these tubules could help give an answer as to why. And they could be something that can be targeted. More work needs to be done but that talk was brilliant.
I first want to explain what a poster session is for those who don't know. Basically you submit a short description of your work and if you're chosen you create a poster (A2) to demonstrate what your work. The more figures the better. You then have an opportunity to stand beside the poster and chat to any interested person who comes along. I want to thank the people who happily stood and answered my weird questions about their work. You're very patient humans. I want to say I went and talked to everyone about their posters (there were 74 by the way) but I hunted out the epigenetics posters and ones that really interested me. I chatted with Tim Fenton from UCL, James Heward from Barts and Neil Slaven from Imperial. I can't really tell you what we talked about because their work is unpublished but needless to say it was very interesting.
All I all it was a really interesting conference, if not a a small bit tiring. My only problem with the conference as a whole was the very small representation from the area of epigenetics. Some speakers mentioned epigenetics (one even had a little data) and there were a few posters on the subject but it was distinctly lacking. I know that's the area I'm in so I am very biased and obviously it was hard enough to fit all the talks they had already into the two and a half day schedule, but it was missed. It may not be the area that solves all the problems (my PhD title will of course be "I cured cancer with epigenetics, everyone can go home now") but it provides a key piece in the puzzle. It's something I hope the organisers will consider for the next Crick International Cancer Conference.
Other than that the 1st Crick International Cancer Conference was worth attending and I learnt some interesting (and terrifying) stuff.
You may (or may not) have noticed that my blog has been all but quiet and desolate the last three months. This is completely my fault and an unconscious act of neglect. There are two main reasons why I have been so absent and hopefully I can give you many excuses to explain these reasons while also updating you on my progress.
The first reason is work. In mid-August I had what's known here as a Late Stage Review (LSR). This is something all Imperial PhD students are required to do in order to complete their PhD. The aim is to present your results and future plans after around 2 years of doing your PhD. You make up a report and do a presentation for two examiners. They judge your work and your ability to complete your PhD and sign you off. Usually they don't fail people (there is always the possibility) and the idea is for them to give you ideas for finishing your thesis and focusing your work. Obviously you want to present as much good data as possible. Hence the absence beforehand. I spent all my time writing the report and generating as much data as I could. All in all the LSR went fine. My work is satisfactory enough to continue. However I am not going to lie I was disappointed afterwards. I thought going in that they would help me narrow down and focus on the more important aspects of my project but instead I was told to "expand more to focus more". I realistically have 12 months left in the lab. This was not what I wanted to hear. I need to add more work to quite frankly an already daunting amount of work. Following from that I wanted to finish up at least one part of my project (validating my significant probes found from the DNA methylation array in another format i.e pyrosequencing). And this you will be happy to know showed...........nothing. Bupkis. Nada. I have negative results. While this is not an epic failure (as my brain wants me to believe) nor is it a triumph. Essentially what this means is I need to go back to my plan and look at the question a different way. And this is when I appreciated the LSR. Yes I needed to do more work but actually it gave me an opportunity to approach my project at a slightly different angle and who knows maybe something will work. I am now in the stages of planning and implementing that work. And who knows maybe in a few months time I will have something positive to talk about.
The other reason I have been absent is I went on a 17 day holiday. I travelled to Rome, Sicily, Dublin and Cork. It was amazing and relaxing. And I didn't have to worry about my project or stress about what I needed to do next. I even slept the whole way through a night for the first time in, well, years. It was incredible and for anyone looking for their next holiday destination I highly recommend Sicily. Get a car like we did, drive around and soak it all in. Mountains, crystal clear water, food. A must really.
And finally on a slightly different note I am taking up some teaching! An ex-member of our lab, Kirsty, is part of a team which has created a brand new innovative BSc undergraduate programme in Imperial. The course focuses on interactive learning and face-to-face teaching. It will be one of the first courses of its kind and hopefully not the last. Part of this course is to teach laboratory skills, called lab pods. I will basically be a part of a team of PhD students in these lab pods who will help guide students through techniques like cell culture and western blotting. Guide being the operative word. Unlike traditional teaching labs where I would show them what to do and answer all of their questions for them, this lab aims to make scientists. So I am really there to watch and provide last resort help. The idea is that the students will get a protocol, read through it and do the experiment. If it goes wrong then they have to figure out why. Exactly like anyone working in a lab would. Besides having a master student, I haven't really taught before so this is a new and exciting experience! Terrifying (especially when you realise you're about 10 years older than they are) but exciting.
And that's what I've been doing. hopefully I will be a bit more dedicated to the blog and I can keep you updated on my work, my teaching experience and also complete a few posts (such as my "What the Hell? - Cancer" series).
Btw I am not quitting this PhD any time soon in case you were worried.
In this edition of "What the Hell? - Cancer" I will going through how cancers are diagnosed. This will hopefully give you an idea about what the different stages of cancer are and how each cancer is graded differently.
NOTE: All my examples will be using breast cancer because it is the cancer type I study and am most familiar with.
Before I begin I want to just clear up the differences between primary tumours, secondary tumours and primary secondary tumours.
Cancer diagnosis is a tricky business. If you read my awareness posts you will see the signs and symptoms of a lot of different cancers. You may have noticed that a lot of the signs and symptoms are generic for example bloating, intestinal discomfort, a prolonged cough etc.
Tumours need to be detected before you can take a biopsy etc. These are the main ways tumours are detected (but not the only ways). Apologies for the explanations. While I learnt all this in my masters (woo nuclear physics) I am no physicist...
Part of what pathologists are looking for when they stain biopsies is structural changes to cells. Cells "adapt" to their environment or from internal cues by changing their structure. There are a number of cellular adaptions which happen normally but can also be due to disease etc.
The next three are characteristic of pre-cancer cells (i.e. structural changes that can lead to cancer):
Change of shape and structure and the number of darkly stained cells allow pathologists to grade tissue samples. Histopathology is based very much on the person and takes years and years to generate enough knowledge to be able to look down a microscope at a purple stained piece of tissue and say "that is a squamous cell carcinoma" (type of skin cancer - don't look up images). There is a lot more to the work of a pathologist but I don't have the knowledge to delve deeper.
Staging and Grading
So what do I mean by stage? Well the stage of the cancer tells you (in general terms) how big the tumour is, if it has invaded locally or systemically and what treatment you should use.
Clinicians expand on this and use a system called TNM. This is "Tumour", "Node", "Metastasis".
As I said before each cancer type has slightly different staging criteria. This is due to the type of organ it is in (some organs are smaller than others so a "small" tumour in a large organ is actually quite a big tumour in a smaller organ). The staging also has to take into account where the tumour invades locally and systemically and if lymph nodes are involved.
In breast cancer:
Other cancers like Colorectal Cancer use additional staging techniques for example Duke's Staging which is very like the numbered system but is marked by grade A-D.
But the above is not the only way to identify the type of cancer! Oh no, like all aspects of cancer biology there is always more.
While the stage/grade will help you with treatment options there are other factors such as location that tell you about the cancer.
For example in breast cancer:
Mutations and Chromosomal Changes
And as always we come back to DNA. There are mutations that are common in pretty much every cancer type (e.g. p53) but other mutations are specific to certain cancers of families of cancers. For example BRCA mutations are associated with both breast and ovarian cancers.
In breast cancer the common mutations used to define cancer types are:
In breast cancer if you have suspected Her2+ cancer a number of tests are done such as Immunohistochemistry (IHC) which detects Her2 receptor levels on the surface of the breast cancer cell and Fluorescence In Situ Hybridisation (FISH) which detects the number of copies of the HER2 gene there are.
There are genetic screening tests used in the clinic for some cancers. In breast cancer there is OncotypeDx which detects 21 genes commonly mutated in breast cancer. This gives you a score. OncotypeDx also has systems for prostate and colon cancer.
Changes to the chromosomes themselves are also common, usually in haematological malignancies.
In chronic myelogenous leukaemia (CML), there is a translocation called BCR-ABL where part of the chromosome where the ABL gene is located (Chr.9) breaks off and switches with the part of the chromosome where BCR is located (Chr.22). This brings BCR and ABL together creating a powerful oncogene.
And you combine them all. You take your image of the tumour(s) with all of the measurements and origin of the primary tumour, you get the pathologists report from the biopsy about the type of cancer it's believed to be, how fast it's growing, what proteins it may be expressing on the cell surface and how aggressive it is and lastly genetic testing to determine the mutations. This gives you a picture which allows you to provide the best treatment plan for that patient.
So a patient could have a T2N1M0 ER+ invasive lobular breast carcinoma, meaning they have a breast cancer which originated from the epithelial cells in the lobes of the breast, the cells show structural changes and it is between 2cm and 5cm across, there 1-2 lymph nodes with infiltrating breast cells, there is no metastasis and the tumour is oestrogen receptor positive. The treatment path for this patient may be surgery to remove the tumour and lymph nodes involved or a full mastectomy. Depending on the surgery, the patient may receive chemotherapy and will more than likely be given tamoxifen (anti-oestrogen receptor drug) for 5-10 years.
It's difficult to describe every type of cancer's staging systems because as I said it really is dependent on the type of tumour.
And that is the basics of it. As always if you have questions please don't hesitate to ask. This isn't all of the information and if you or someone you know is affected by cancer they should ask their attending physician to explain their diagnosis to them (cancer is so nuanced).
Hopefully in the next month or so I will put up our penultimate "What the Hell? - Cancer" blog post on Cancer Treatment!
For more information check out CRUK's website https://www.cancerresearchuk.org/
On Sunday the 25th June I participated in the Chiswick 10K Fun Run! I am not much of a runner but I did walk very briskly! I also had the great opportunity to say a few brief words before the event to thank all the participants and tell them where their hard earned fundraising goes.
I had a great time walking around Chiswick! It was fantastic to see the local community come out and run/walk against cancer. There were many stalls included CRUK science demos, face painting, a DJ from the local radio station, a burger van and more! The great volunteers and organisers put a massive effort into making the event fun and engaging!
While the weather wasn't sublime it was perfect for running/walking in! The volunteers along the route were great at giving some encouragement, water and much needed directions! I was even given a jelly baby along the route which made my day! After 6km I was dying for some sugar!
I love doing these volunteering events for CRUK, it gives me a lot of joy to be part of a team spreading such a positive message about beating cancer. And whether your event has 5 people or 5000 people, it can create so much awareness about cancer and about where money fundraised goes. I'm just glad to be a part of it! Even though I'm dying today after it!
Just as an update, I did a little interview with the Chiswick Buzz at the end of the walk! If you want to see more about the day and also my small little part check out this link:
I'm at the end!
On Thursday 1st June I was asked to present to a group of incredible volunteers for Cancer Research UK. Each year the Royal Family hosts garden parties at Buckingham Palace to show their appreciation for people who have made a positive impact in their community. It is a great honour to be invited. This year CRUK was allowed to send a small number of volunteers to the party to say thank you for being so great! I went to a pre-party gathering in the CRUK headquarters in Angel where I did a brief presentation about how I came to be in London, why I study cancer and the background to my project.
It was a pleasure to meet these incredible women and to chat to them about my work! Please enjoy watching the video of my presentation.
Warning: I say "em" A LOT! Also I exaggerated the number of women participating in the EPIC study - it's half a million.
Disclaimer: the opinions in this presentation are my own.
Thank you to Becky from CRUK for videoing this for me! 😊😊😊
Today I am starting a multiple part “What the Hell?” series on the subject of Cancer. There are many different aspects of cancer biology from what cancer is to how we diagnose and treat cancer and how we can prevent cancer. These posts will be a bit longer than usual so bear with me. I want to cover all of these subjects in enough detail that you as a reader can walk away with a few answers and maybe also a few questions.
HEALTH WARNING: These posts will be long. There is a lot of text. Feel free to read a little and come back.
To begin: If you don’t take anything away from these posts, I do want you to take away one VERY important thing. Like all facets of life, cancer is not black and white but a vast, daunting expanse of grey. As unique you are from the next person, cancer is unique to the person who has it. While cancers fall into “families” based on location or genetic similarities (e.g. breast, lung, etc.), each one has it’s own characteristics that make it unlike other cancers in the same “family”. In the simplest way I can come up with, cancer is a collection of your own cells that have gone mad. And that madness is unique to you.
1. Solid Tumours:
Blood (liquid) Cancer:
As I said cancer is your own cells that have gone mad. In more scientific terms, there a certain number of characteristics (or "Hallmarks") that define cancer set out by Hanahan and Weinberg originally in 2000 and later updated in 2011. Figure 1 shows the classic “Hallmarks of Cancer”.
The first three Hallmarks are very related and focus around signalling. In a nutshell, cancer cells need to continue to grow while preventing the cancer cells from dying.
(1) Sustaining proliferative signalling
Normal cells carefully balance growing with not growing. This process maintains the right number of functioning cells. Growth signals are given when more cells are needed but are shut off when no cells are needed.
In cancer, the growth signals are constantly active. This means the cells are constantly growing and dividing. They can do this in a few ways:
(2) Evading growth suppressors
As I’ve said before growth is tightly regulated. There are lots of signals, which inhibit the growth of cells. Cancer cells actively prevent these signals from preventing growth. This can be done by:
(3) Resisting Cell Death
Cancer cells do not want to die. They want to keep growing and dividing. Normal cells have a few ways of dying which controls cell numbers but also prevents cells with DNA damage or bad mutations from becoming cancerous. These pathways include: apoptosis (programmed cell death), necrosis (un-programmed cell death) and autophagy (where the cells breaks down part of their own cell structure to survive). Cells receive internal (DNA damage, shortened telomeres etc.) or external signals (from other cells) which tell the cell "Listen...I think it's time for you to pop your clogs".
Cancer cells deregulate cell death pathways by shutting down the pathways that lead to cell death and increasing the pathways that inhibit cell death. This means no matter how many internal or external signals the cells get to die, the cancer cell will ignore them and continue being a cancer cell.
In summary for the first three Hallmarks, cancer completely re-wires all the signalling pathways in the cell to promote their growth and division while ignoring any prompts to stop growing and die.
The next three Hallmarks give cancer cells a growth advantage and allows the cells to move around the body.
(4) Enabling replicative immortality
Cancer cells are immortal. This means if they were allowed to keep going uncontrolled, they will grow and never die. Normal cells have a life cycle. They grow and divide and if they don't receive premature death signals, they keep going until they have a "natural death". This is controlled by things called "telomeres". These are long stretches of "TTAGGG" base repeats that sit on the end of chromosomes, like the caps on the end of your shoe laces. They protect parts of the chromosome from being lost during cell division. Every time a normal cell divides the telomeres shorten a little bit. When they become too short the cell reaches a "crisis" and dies. This is a natural death for a cell.
As a cancer cell starts to become malignant, the telomeres still shorten. However when the telomeres reach "crisis", the cancer cells turn on an enzyme called "telomerase". Instead of losing telomere repeats during every cell division, telomerase now adds telomere repeats. This essentially prevents the cells from going through natural death and makes them immortal.
(5) Inducing angiogenesis
All your cells need a healthy blood supply. Blood vessels bring oxygen and nutrients to cells and take away all the bad rubbish like CO2 and waste. Your organs are covered in blood vessels to make sure every cell has access. When your cells have a good supply of oxygen it is called "normoxia" (basically normal oxygen). When your cells are starved of oxygen it is called "hypoxia". You don't want your cells to become hypoxic because it kills cells.
As a tumour develops it wants to grow near a good blood supply. But that may not be enough so cancer cells can grow their own blood vessels. This can be part of the early development of the tumour or because the tumour is so big the cells in the middle are all hypoxic. Cancer cells grow blood vessels by sending out messages to their local blood vessels (for example VEGF-A or FGF). This causes the blood vessel cells (endothelial cells) to branch off from the main blood vessel and grow towards the tumour. Tumour blood vessels are pretty badly made (figure 3). They're leaky and are pretty weak with large gaps between cells instead of a smooth blood vessel. While inefficient, they work well enough for the tumour to get the oxygen and nutrients it needs.
(6) Activating Invasion and Metastasis
The final established Hallmark of cancer is the ability of cancer cells to spread.
Cells live together in organs and tissues. But there is a lot of stuff outside the cells which provide support and structure for the cells (for example collagen). This is called the "extracellular matrix (ECM)". The ECM is pretty stiff. This prevents cells from moving around too much, giving them an anchor to other cells.
In cancer, cells want to occupy the area around them. Essentially they "invade" into the extracellular matrix. They do this by:
Invasion and angiogenesis (making blood vessels) allow cancer cells to metastasise. Metastasis is basically cancer cells moving from one part of the body to another. The primary tumour (the original tumour) forms a secondary tumour (the new tumour) in a different part of the body. The old tumour and new tumour share very similar characteristics. Cancer cells usually don't just move anywhere but pick specific parts of the body to call home based on the preferences of the primary tumour. For example Breast Cancer usually spreads into the chest cavity, liver and brain. How cancer cells pick the new sites is relatively unknown.
Once the cancer cells invade into the ECM they can move towards blood vessels. When they're in the blood stream they surround themselves with immune cells. This is the not only to avoid being killed by the immune system but also to withstand the pressure of blood vessels. If the cancer cell did not protect itself it would be torn apart very quickly. The cancer cells can travel vast distances throughout the body. When a cancer cell picks a site for the secondary tumour, it leaves the blood and starts to break down the ECM at the new site to create space to grow. The cancer cells also transforms back into epithelial cells (MET).
Even though primary tumours are constantly shedding cancer cells, only 0.1% of cancer cells broken off from a primary tumour actually form secondary cancers.
Figure 4: Tumours release signals into the microenvironment to create a path for an individual cancer cell (purple cell) to get to blood vessels. Once in the blood vessel, the cancer cell hijacks immune cells to protect itself (blue triangles). The cancer cell can travel vast distances all around the body. The cancer cell then leaves the blood vessel. It breaks down the microenvironment around itself to create a space to grow into a new tumour.
In 2011, Hanahan and Weinberg updated their Hallmarks with four more, two Emerging Hallmarks and two Enabling Hallmarks.
(1) Deregulating Cancer Energetics:
Cancer cells use a lot of energy compared to normal cells. This is because they are constantly growing and creating proteins, lipids and nucleic acids. The primary source of energy cancer cells use is glucose. To get the most bang for their buck cancer cells do something called the "Warburg Effect" (figure 6). In normal cells energy is made using "oxidative phosphorylation". While you get a lot of energy per glucose molecule, it takes a long time. Whereas another form of energy making, "glycolysis", is extremely quick. You don't get as much energy but cancer cells are more concerned with speed than efficiency. Glucose can also run out quite quickly so cancers have adapted to use other sources of energy for example lactate.
(2) Avoiding Immune Destruction:
Your immune system is pretty sensitive to foreign invaders. All cells have signals on their cell surfaces (called antigens). Immune cells produce antibodies which bind to these signals. If the signal comes from your own normal cells, the immune cell leaves it alone (kind of like a friendly handshake). If the immune system doesn't recognise the antigen then it attacks the cell.
Cancer cells are not normal and do express antigens which immune cells do not recognise. To avoid being destroyed by immune cells, cancer cells express the "normal" antigens to trick the immune cells. Cancer cells can also hijack the immune system. By sending signals to certain immune cells they can transform them into pro-cancer immune cells. When an anti-cancer immune cell tries to destroy the cancer cell, the pro-cancer tumour cell comes in (kind of like a body guard) and suppresses the anti-cancer immune response.
(1) Genome Instability and Mutation
I think I've established that cancer cells have lots of mutations which helps them grow and divide and generally be cancer. But there are certain mutations and genome instability events that are indicative of certain cancers. Cancers go though a transformation pathways from normal cells to malignant to metastasis. Each type of cancer has a sequence of events that gets them to that stage. For example figure 7 shows this sequence in Colorectal Cancer.
(2) Tumour-Promoting Inflammation
Inflammation occurs naturally in the body, usually in response to an injury. Inflammation is the response of the immune system to injury or damage. As I've said before, cancer cells hijack the immune system to protect themselves. But a side effect of this is the immune cells create a highly pro-cancer environment. Inflammation leads to the release of a wide range of growth signals, it promotes new blood vessels to grow amongst other things. In essence inflammation is the perfect environment for a tumour to grow.
So those are the main Hallmarks of Cancer. While these were defined in 2000 and 2011, how cancer cells do any of these processes is still largely a mystery. I will talk later about how we can target Hallmarks of Cancer to kill cancer.
My name is Caitriona and I am a PhD student at Imperial College London, UK.