Dr. Victoria Forster

So I’d just like to say thanks again to everybody for attending this session.  It’s always a real privilege to talk to our families and our patients.  So, it’s wonderful to be invited to share my work with you today – I really appreciate your time.

I’m going to talk about something completely different to Vanessa.  I’m actually going to tell you exactly what we do in the lab with a CMMRD patient sample. Many of you will have donated your samples, or will have donated your children’s samples.  And, many of you who are physicians and genetic counsellors will have worked with patients and families to coordinate getting us samples from all over the world. First of all I just want to say thanks for that and I’m going to tell you a little bit about what we do here.

Vanessa talked about the consortium, and one of the things that the consortium does is collect patient samples. So, what kind of samples do we get through this consortium?

Well, to me, as a lab-based research scientist, there are two different types.

One type of sample is the type that we use to just extract DNA, which is really important for identifying mutations in tumour and figuring out which mismatch repair genes are mutated. We can get DNA out of all sorts of things.  For example, we can get microscope slides with bits of tumours and we can scrape those off, and people like Lucie do a wonderful job of extracting DNA out of them.  Samples that we get for that kind of work don’t have to be alive.  DNA is a really stable molecule and you will probably have seen Jurassic Park and things like that, where they get DNA out of the dinosaur bit in the amber and things. Now, we don’t use dinosaur samples, but we can sometimes get DNA out of samples that are many, many years old. So, that’s one type of sample that we use.

But, the type that I really use a lot is “alive” tissue. This is tissue that patients get removed as part of an operation. So it could be a tumour sample, or it could be a gastrointestinal polyp, or it could even be a tiny sample of their skin. And, we actually keep that tissue alive whether we collect it here in Toronto, or anywhere in the world.  We actually try and use it and keep it alive in the lab.  This sounds a little bit like Frankenstein, but trust me, it’s not quite that bad.

For my work, I mainly use two types of live tissue. One is called a skin fibroblast. Fibroblasts are these kind of small star-shaped cells that grow all over your skin, and also in other parts of your body like your colon and your intestines, for example. They are very abundant.  They grow really well. They’re like a scientist’s dream because they are very unfussy and the food they need is not very complicated. So, we sometimes get small samples, very, very tiny samples (I think they are about a millimeter across) of skin, and they contain these fibroblasts. I’m going to tell you about what we use these for today.

The other type of sample I use a lot of is intestinal tissue. Many of you will know that as part of our surveillance protocols, our patients have colonoscopies to identify any polyps. Therefore, I get samples of polyps, I get samples of tumours and also sometimes tiny chunks of healthy intestinal tissue, too.

For the rest of my presentation I’m going to tell you about what I do with both of these types of samples.

If anybody has ever waited for a package through Canada Post or Purolator, or something like that, you’ll know that it’s a very stressful experience. Well, imagine that what you’re waiting for is very fresh human tissue and if it doesn’t arrive in a day or two, you can’t use it anymore and it’s pretty much useless for experiments. You can see Vanessa and Ayse, one of our other colleagues, here and there is them coordinating a shipment of fresh human tissue from somewhere in the world. The majority of our samples come from North America for fresh tissue, just because of logistics, because we need to get them either on the same day, or ideally overnight otherwise the cells don’t survive very, very well. But, we have actually managed to get samples, I believe, from Israel and also Europe and possibly a couple of samples from Asia as well. As you know planes, if everything works on time, can get us tissue from anywhere in the world within about a day, which is remarkable.

Skin fibroblasts are the easy ones for us to receive.  They are sent in something called a tissue culture flask, which I actually have one here in my house. Please don’t ask me questions about that, it’s a little strange. But, basically, it looks like this, and the fibroblasts grow along the bottom of this flask and they just stick there. If the people who are sending us the skin fibroblasts just fill up this flask and seal it really, really well, they can just ship it at room temperature to us in a polystyrene box. Normally, by FedEx or something you would use to ship an Easter present or a Christmas present to a family member.

Colon tissue is a bit more complicated and what causes most of my stress (and Vanessa’s stress as well), hoping that it gets here in time. For colon tissue, we can’t freeze it because that would kill the cells that we want and we can’t send it at room temperature because that would also kill the cells that we want.  We have to keep it cold. So, it’s sent it’s sent in special tubes in special liquid medium that we send out to everybody who is going to give us a sample. And then, it’s sent on ice packs in a polystyrene box and we’ve got about a day or two to get it before it doesn’t become very useful for us.

So, why colon tissue? Well, as I said, we get different types of colon tissue. We get polyps, we get tumours and we get tiny bits of healthy tissue as well. The great thing about colon tissue for a scientist like me is that it’s full of stem cells – absolutely full of stem cells. There is a really good reason for that, which is that every time we eat or do normal stuff, our stomach lining gets really, really stressed out. You will have all have heard of things like stomach acid and enzymes that we use to digest our food.  Well, all of these things are incredibly stressful for the inside of your intestines. So we’re designed to actually regrow our inner lining of our intestines’ every few days. These stem cells actually allow us to do that.

You can see here, I’ve got a little diagram of what the surface of your intestine actually looks like, and it’s covered in things called crypts. They kind of go up and down like this to increase the surface area for absorption of nutrients, water and other stuff from your food. At the bottom of each one of these crypts is a little pocket of stem cells, which are ready to kind of grow and replace anything which is lost.  Also, when some intestinal cells get damaged, it just immediately gets replaced.  It’s these stem cells at the bottom of these crypts that make them really useful for me. This is because they can actually regrow an entire mini-colon in the lab.

So, how do I do that?  Well, I take the crypts off using a combination of different chemicals. We don’t need all of the tissue, we just need these crypts and these stem cells. Then, I actually put them in these tiny little jelly domes.  If you’ve got good eyesight, you can probably see these here. They’re about, 7 or 8 millimetres across and they kind of look a bit like a cookie.  They’re kind of raised in the middle, and when they hit the outside they kind of stick to the surface. So, in each one of these little clear jelly domes is about 200 of these crypts from the intestine. After I’ve done this, and it’s set hard, I put a layer of liquid media on and I leave it for a couple of weeks. After a week or two, it looks like this. Each one of these little structures you see here, whether they be the little kind of twisty blobs here, or these more cystic-like things, came from an individual stem cell that I took out of that sample that we received. I hope you can see my cursor but if not, it’s at the bottom left of the screen. You can see that some of them look like little tubes. So, these stem cells are incredible because if you give them the right environment, they will actually grow into tiny little intestines, basically.  Which, I mean, every time I do it till impresses me because it’s so cool.

We have, I think, colon organoids from maybe 10 or 15 patients now in our biobank so luckily, I don’t have to keep these growing constantly because they’re very demanding. It does allow me to take days off sometimes and have holidays, which is great. We actually are able to freeze them down. First of all, in our -80 freezer. You’ve probably heard lots about those in the news at the moment, with regards to COVID vaccines. They look a bit like this. Then we put them in liquid nitrogen and we can store these forever.  We can defrost them and about half a day after we defrost them, they will start growing again.

We have this biobank now and we are asking a number of questions. First of all, we are trying to figure out how CMMRD patients get colon tumours. As I said we receive healthy tissue sometimes and we can make organoids. We also receive polyps, and we receive tumours. So, we can look at the different organoids made by these different samples, compare them, and see what’s happened between these stages. We can also try and test drugs to treat CMMRD colon tumours. We can then also try and test therapies to prevent the development of these tumours in the first place.

I’ll talk a bit about a few more questions we’re asking at the end of my talk.

The fibroblasts may not look as cool but actually, they have a lot more potential, I would say. We’ve already talked about how we get these and how we grow them in dishes. Here are some on the right, here, which I’ve dyed blue. They don’t, to me, look very exciting because I’ve seen them many, many times over many, many years. You might ask, why are you getting skin biopsies? Skin cancer is not something that we see very frequently at all in CMMRD individuals. I don’t know if we’ve even had any.  But if we have, it’s very, very rare. So, why the heck are we collecting skin cells?”

Well, as all of you will know, the mutations that cause CMMRD are the same in every cell in the body. So, whether we take a brain sample or a skin sample, or a colon sample, the mutations in PMS2 or MSH6 or MSH2 that cause CMMRD are in there.  And so, these skin cells also have the same mismatch repair mutations.

So, what the heck do we do with them? This is the bit which I still struggle to get my head around sometimes. You can actually take these skin fibroblasts and you can reverse engineer them into stem cells. And then, we can do whatever we want with those stem cells. We can make blood cells, colon cells, and brain cells. It’s a long complicated process as to how it’s actually done. But essentially, we use viruses to persuade these skin cells that they should become stem cells again. And then, we have stem cells that have the same genetic mutations that are in those patients, but that we can do various things with.

You can see here, they look very, very different to the fibroblasts. These big black dots here are actually the DNA in the stem cells. In some of the cells, there is actually two blobs of DNA. That shows me that the stem cell is actually dividing. So, that’s kind of cool.

I’ll talk to you about a couple of different tissues that we make from these stem cells.

So, we can make colon organoids again.  Even if we don’t have a colon sample from a patient, if we have a skin sample which we can make stem cells out of, we can make mini-colons, and see how those colons behave with those genetic mutations.

This is one of my favorite-ever mini-colons that I have made. It just looks so interesting because it’s got loads of different little tubey-bits, and bulgey-bits. Not only can we make these, we can have a look at them and we can study them. Here, on the right, you can see a couple of dyes that I’ve put on. The one on the far right, which is a brown dye, actually identifies a mismatch repair protein called MSH2. We can see in this particular one, it does have MSH2. This is a control organoid that I use for my experiments. It doesn’t have mismatch repair deficiency. And the purple stuff is basically just a kind of pretty stain that shows us that the cells are where they should be.

The thing that I think I do which is most cool is that we can make mini-brains from these stem cells that we get from skin cells. It sounds like science fiction, but trust me, we do it on the 17th floor of the SickKids Research Tower. This is probably my favorite thing that I’ve ever done in the lab. They’re absolutely tiny to start with. On the left, the kind of grey picture, you can see there’s loads of bubbles coming out of them. This is about two weeks after of we start growing them from stem cells. A few weeks later, the little spiky bits that you can see on the picture in the middle are actually neurons starting to form and coming out of the mini-brain. And then, in the top right, these are the mini-brains entirely formed and they are about 2mm across.  They’re tiny.  Tiny little white blobs.

So if a child has never even had a brain tumour at all, with a skin sample, we can see how their genetic mutations might behave in brain tissue. Because, of course, we don’t want to be taking brain tissue from healthy patients. We can’t do that, and we would not want to do that. But with this, we can study what might happen to their brain tissue should they ever get a tumour, or whether it’s likely, for example.

And so one of the questions we’re asking is simply: do our mini-brains with CMMRD eventually get mini-brain tumours?

This is a picture which essentially allows us to look at the structure of the mini-brains. We essentially slice it in half, and look at the surface of the mini-brain. You can see, there’s loads of different types of structures. It doesn’t look exactly look like a normal brain or a brain scan that you would see – these are not that advanced. But you can see, there are different circles there and these are things that look like little holes in the mini-brain. We actually also see in normal brains. They’re called ventricles in human brains and they allow for nutrients to be exchanged in the brain. You can see on the left the actual size, but our microscopes allow us to zoom in much further and look at even individual cells in the mini-brain.

This is a little section of one of the brains. Each one of these blue dots, here, is a single cell. We can see that they’re at a different structure. This is one of those holes that I talked about, which we’re really interested in because there is some thought in the medical field that a certain type of brain cancer might start around these holes. We can see, in our case, whether that is the case.

We actually do some really exciting work with a group in the UK. The group in the UK uses a laser to dissect bits of our mini-brains and then have a look at the DNA sequence of those bits to see how it compares to other bits that don’t have CMMRD and other mini-brains that don’t have CMMRD.

Just before I end, because I think I’m a little bit overtime and I don’t want Lucie to get upset at me.

We’ve got a load of ongoing research projects that are using these organoids to actually answer real questions that make a difference to people like you.

So, we want to ask the question: why do some of our patients get loads of brain cancers and some just get colon cancers and no brain cancers at all?

We are asking, why some patients get loads of colon cancers and polyps and then some get none at all?

And we’re doing another really exciting piece of research with another group in Europe actually investigating whether it might be dependent on a combination of normal gut bacteria, which all of us have, but also whether there are certain types of gut bacteria which might predispose some people with CMMRD to getting colorectal cancers and polyps more than others.

So, we’re really using these organoids in as many ways as we possibly can to answer loads of different questions.

And, as Melyssa Aronson said, for example with EPCAM, there are questions about whether that causes CMMRD in all tissues or not. These organoids can perhaps, help us answer those questions as well. So, they’re really exciting

I just want to say thank you to everybody else in the lab who has helped with this work. Cindy Zhang, in particular, has done a lot of this amazing work.

We are a really, really good team and we work together really well. Sometimes when the colon organoids need feeding, somebody will agree to do it for me so I can have a day off in the middle of these big experiments.

I guess I just want to end by really encouraging you to ask as many questions as you want. This session is for you, there are no stupid questions at all. I’m more than happy to answer anything about what I’ve just shown you today and I really hope you feel comfortable asking, asking loads of questions.

Thank you so much.