Science Lite — Using Zebrafish to Study and Model Cancer


[music] So as Jody said, my name is George Eisenhoffer, I’m a postdoctoral fellow in the Rosenblatt lab and I’m funded by the
American Cancer Society, and today I just wanted to tell you a little bit about
how we use zebrafish to study of model cancer in the lab, and as Jody said, we
think this is kind of a new way for and one of the best model systems to study
cancer currently, so, and I’m going to hopefully try to convince you of that
today. So I don’t really need to tell this audience too much about cancer but
what I thought I’d talk to you a little bit about today is just the
generalized version of cancer, that the way we view it, and I think that this
topic needs little introduction mainly because everyone in this room has had
probably a close friend or family member that’s affected by cancer and this is
also why a lot of us go to such great efforts to either study this disease or
for the fundraising efforts to raise money for those types of research. And
despite about 100 years of research so far we actually know very little, still,
about these initial steps of cancer. How do normal cells go from just sitting
static to actually piling up and accumulating overtime. Now we know that
they actually start out normal, but over time we know that due to environmental
or toxic insults they start to acquire genetic changes over time that actually
cause them to pile up. And after they start piling up, this would be the
initial tumor formation, here. Eventually they can actually leave this primary
site and it’s called metastasis. This is actually the deadly portion of the
disease, when they can actually leave the primary tumor and go and set up shop
somewhere else. Now, in the Rosenblatt lab, we were very
interested in this very early initial steps of actually how this forms. And so
when I started my postdoctoral fellowship through the American Cancer
Society in Jody Rosenblatt’s lab, we actually set out to understand these
very initial, early steps, and we think that this is a very simple problem that
I, we like to term ‘a personal space issues’ of these cells. And so to do that I think
I’ll just lay out a little bit about personal space. And so first off,
we know that there’s just basic zones of human interaction and this is design,
defined by how familiar you are with the person. So in public spaces we stand
approximately twelve feet apart from each other about how far I am from you
now. In social settings most of us stand three to four feet
apart, when you really know the person you can stand one to two feet apart, and
when you’re intimate you’re standing right next to them, but just normally in nature
people tend to sit approximately three to four feet apart when they’re
just sitting static. Now some humans have combated some of, found some ways to
combat personal space issues, so this gentleman here found something to keep
people away from them, but we’re really interested in when this fails, how does
this, what happens here? And so we like to think of cancer as a failure of cells to
actually observe their neighbors in this personal space issue. And so I think that
the, we can all appreciate the first panel here, and that personal space
issues can arise from cultural differences. I’m sure everyone this room
probably knows somebody that stands a little bit too close to you when you’re
talking, you know, and you know, in the old country they did, some places don’t have
personal space. This can also be if two cells get too close to each other they
can start to be aggressive towards each other. I think baseball exemplifies this
very nicely. And finally, if this balloons out of control, you can actually have a
scenario where people start piling up on each other and the system completely
breaks down, and that is just shown here, it’s the subway. But what we really were
interested in, does, how does this concept of personal space work at the cellular
level? And so, as the old saying goes, ‘the devil’s in the details’ and he’s here to
know about the details, so that’s actually what I’m here to tell you a
little bit about today. So one thing that we have learned about many of these
different types of cancers, the breast, lung, and prostate, is that despite them
being in all different tissues they arise in a very similar way. They acquire
genetic the cells, acquire genetic lesions, and they actually start piling
up over time. Now one thing that these tissues all have in common is that they
are all, arise from epithelia. So epithelia coat our body and our organs and provide an essential barrier from the outside world.
That is just shown here where we have oral epithelia in our mouth, epithelia
coating our kidneys, our stomach, basically every organ in our body. Now
80% of all the cancers actually form in carcinomas on your epithelia, and this
could either be breast, lung, or prostate, but the process of carcinoma formation
is actually similar in all these different tissues. So when I came to
Jody’s lab, we actually set out to understand how epithelial cells
themselves are the ones that have problems with these personal space
issues, and to do that we wanted to take a step back and just look at the very
essential processes of how cells even determine how many, count the numbers
of the, of themselves in the tissue and assess their neighbors. And in doing this,
we wanted to look at just the very basic balance of cell birth and cell death and
that is shown here, and this is an epithelial monolayer and culture, where
you have a dividing cell, here, so that’s gonna make two different cells and
that’s called cell division, and this other cell, marked by this red spot here,
is dying, and that’s just the cell death. Now, in our body, approximately 100,000
cells are produced every second by mitosis, and an equal number die. So just
while you’re sitting here listening to me, this process is going rampant
throughout our body. Now you can imagine that this has to be very tightly
regulated because it’s happening on such a dramatic scale, every few seconds. And
so we really wanted to set out as how this balance is maintained and what
controls this? So what happens if we get too much cell division? Well then you
start, this is actually, if cell division outpaces cell death this is actually how
you get tissue overgrowth, and we think the initial steps of tumor formation. On
the contrary, if you have too much death and actually not enough proliferation,
and you’re not sensing that your neighbors are leaving, you can actually
start to get holes in your epithelium and compromise barrier function, and this
is a common problem after chemotherapy where a lot of the different cells in
your body are actually hit at once with a nasty drug and a lot of them start
dying. So we just want to understand the fundamentals of actually how this was
working, and we put forth a model recently that physiological cell
turnover is actually just driven by just mechanical cues. So most people who’d
actually looked at this have hit cells with either a nasty
chemical or some kind of other treatment, this UV, to actually induce death and
watch how this happens. When I came to Jody’s lab, we chose just to actually
look and see what happened to cells normally. How are they just normally being
born and die within a simple population in the absence of any nasty chemicals?
And we think this is driven by mechanical cues, such that if you have the
normal cells, and they’re just sitting there, if they become too crowded, too many people get in there, kind of have an instant bouncer mechanism, so one of the cells is actually just kicked out of the tissue and we call this crowding-induced extrusion. On the flip side, when you have too few people, the cells can
also sense this and they start dividing and make more of themselves to maintain
overall numbers. So we think that in the absence of any other stimuli that this
can just be driven by a normal just turnover. But what we really want to understand
was the fundamentals of cancer from the very beginning of the tissue all the way
down to the genetic level, and that is just shown here, where we have an outline
of the stomach in the colon with a very simple epithelial tissue. When you zoom
in and actually start looking at this at the cellular level you have nice simple
columnar epithelial cells. And when you look inside the nucleus of these cells
you see nice DNA that is packaged in a different way, this is actually how the
information is transmitted from cell to cell and generation to generation. So we
are actually interested in how regions within these are changes, genetic changes,
but the DNA change this information and how it’s perceived within these cells
and what that actually means for the tissue. So how do we actually go about
doing this is the big question. Now, there’s a lot of commonly used model
organisms to study human disease, and one reason that we use these model systems
is if these all these different animals are actually very much simpler than
ourselves and it provides a nice way to model this in a much easier context to
understand. Now, also, one other thing that these animals have going for them is that many of them have exaggerated traits that we have selected
for in the lab. Now on the top panel here, I have all-vertebrate organisms, besides
these are cells, they actually came from humans, so there are cultured in that dish,
but all these animals have vertebra and these animals down here are
invertebrates. They have all shed light on different biological processes.
The fruit fly, the nematode, have been essential for understanding genetics and
how information is passed on from generation to generation. Yeast has been
important for understanding how the DNA is packaged and a transcription and how
these, this information is encoded. While the mouse and the zebrafish have
actually helped us to understand the cell and tissue level, what happens when
these processes go awry? Now, people also study this in human cultured cells but it is
important to look at it in the context of the whole animal. Now, while the mouse has been very important to be able to do this I’m
gonna try to convince you today that the zebrafish is really the way to go
forward. And the reason we feel that is is because we can use the common aquatic pet, so this is the same one that all of you see at the pet store, that you’ve been
there, and it’s just, we use the species, Danio rerio, and the reason we think this is a really important model is that for the adults
they’re very small, they’re very easy to maintain. They have a low cost to house and maintain over time. Important for us, they generate
hundreds of fertilized eggs per week. Now normally in the wild these eggs are just
lost and eaten by the other fish, but we can actually put these to use and
actually study them. This also provides us the way to study development in
adult stages and finally, these guys can regenerate both their fin and
surprisingly their heart, you can cut off about approximately 20% of their heart
and they can regenerate it very rapidly in a scar free manner, which is amazing.
The most important for the talk today is that these animals actually sporadically
form cancers in the wild and in the laboratory setting, so just like humans
do, as they start to get older over time these animals can start to form cancer. So we can make them form cancer by performing genetic tricks, but these
actually just happen normally. Now, one thing I haven’t told you about
is, I told you how great this animal is, but what we actually look at here,
normally in the lab, is the embryonic development or the larval stages of
these animals. And so these animals, I told you, they generate, the adults
generate approximately a hundred of these eggs right here a week. So you can see
this outer shell right here is called a chorion, or an eggshell, and the animal
developing in there, this is a 24-hour embryo, so this is 24 hours after the egg
was first fertilized and you can already see that the eye is developed, there’s a nice
spinal cord, the brain is right here, and this is the yolk. This is uh, this is 24
hours and this is 48 hours, which we call the larvae, and you can see the heart
is starting to develop there, and the yolk is now extended out, also has the eye, and
these black dots that you’re seeing on the side of the fish are the pigment
cells that will eventually give rise to the stripes in the adult fish, and this
will become important later when I start telling you about, more about skin cancer
and melanoma. Now, importantly, these, I have told you, we have both embryonic and
larval stages, but as, hopefully that you can appreciate these animals are very
transparent, so you can almost see through them, despite these pigment cells,
but in these early stages you can watch all the cells actually moving around in
there, so this actually allows us to visualize the cellular movements and
processes during development of these animals and how this is contributing and
sculpting this animal. And what I’m going to tell you now is the ability of these
animals allows us to be able to perform genetics studies very rapidly. This also can be done on the mouse but it can take years, whereas in the fish we can do
this on the order of months instead, so everything is dramatically sped up. And
so this is the whole system when you weigh it out, where I’ve laid out for you,
that we have a transparent embryo that allows us to see this and visualize
all these events happening very early on. From the time that the egg is first
fertilized, about three months you can actually have
adult fish that allows us to study cancer formation in the adult, but these
are also small and easy to maintain. I told you they generate approximately a
hundred to 200 progeny a week, which allows us to do a lot of different genetic
studies that we can also perform large-scale genetic and chemical screens,
and I’ll talk more about the chemical side of things, how we use these to
identify new drug compounds here in a little bit. But importantly, one major
reason that we use the fish is that there’s actually been generated a
high-resolution genetic map of the whole genome of these fish, and it’s actually
been shown that they actually have conservative relationships of the actual
genes in the DNA between zebrafish and mice and humans. So that means that
things that we find in the zebrafish can be applied to humans and that many of
the disease, many of the different mutations that we found in zebrafish
cause changes, morphological changes in the animal that will appear very similar
to human disease states. So we feel that we can use this as a proxy to really
learn very rapidly what’s going on in different human disease states. So what
does this really look like in the lab? You guys have all seen these fish,
probably in the pet store, but what does it look like when we start working with
them? So this is just them sitting in a basic tank, we could keep approximately
20 of them at any given time in the tank, and you can see here that we just have
rows and rows of these tanks, but what I hope you can really appreciate is that
these are all tanks and there’s two rooms of this, so we have a nice very
centralized zebrafish resource here the University of Utah,
with over 15 different labs studying zebrafish and utilizing all this space. Each one of these different tanks is, contains a different genetic background. So, either a different genetic mutation or transgene with a different tissue
label that allows us to study these fish. So you can hopefully appreciate the
scale of what is going on here, down in this centralized zebrafish resource center. Now I told you that these adult fish start to lay eggs. So what does that
look like in the lab? Some of you may have seen us walking around with these
blue dishes, down walking around in the hallway, and that’s actually what this is. You have approximately 200 eggs in this dish. The blue component to the media as an antifungal component, so if you’re wondering what
the blue is, that is what’s in there, but when you see us wandering around the
halls that’s what we’re carrying. But the beauty is is that we can actually just
throw these eggs right underneath the simple dissecting microscope and watch a
lot of these processes happen over time within the, during development of the
fish. And that’s what I’m going to show you here. So this is an egg that’s just
recently been fertilized and I’m gonna play it for you, and it’s gonna loop back
over, but what you can watch is that the cells went from 2 to 4 to 8 to 16 and
then they start getting to where we can’t count them anymore,
but all this is happening, this whole movie happens over the course of
approximately 20 hours. So you can see all the cells are still dividing here,
this is called epiboly, where it starts spreading out over the side of the fish,
and you’re gonna see the head develop up here and the tail start to develop out
here. You’ll see the eye and the brain form approximately right here, and a
spinal cord start to come up right here. Now this is, the reason that these fish
are very important is that they develop externally, we can watch all this happen,
whereas opposed when this happens in humans it’s all happening inside the
mother, and this allows us to start figuring out how these movements
actually contribute to the formation and sculpting of a vertebrate embryo over
time. And I’m just gonna let it finish playing because I really like this movie, too. I think it’s very easy to appreciate what’s going on with the fish. But I do want you to be able to recognize that it does have some very
complex structures, much like we have in humans with a complex brain, eyes, and
spinal cord. Obviously we don’t have a tail, but initially in development you
have a similar structure to the tail. It develops very similar. Now, over time the
zebrafish have a rapid generation time. So I just showed you this beginning
fertilized egg stage, but if we follow this out just a little bit further, you
can see by 24 hours it actually already starts to look like a little fish. Now if
we go out by 5 days, this is the larval stage that I was telling you about,
and actually some of the more complex structures such as gills and other
things are starting to be formed here, and over the course between five days
and three months we actually get an adult zebrafish. And this bottom one is not to
scale, although the other ones are very close. So this actually allows us to
study the complete development of these animals in a very rapid time period. Now,
other researchers have made use of these, of this rapid generation time, to do
genetic screens, and I hinted about this a little bit, but I want to kind of
highlight the importance of this because I really think that this is where
zebrafish excels and I really want you guys to understand this portion. So back
in the early 90s two large groups, one in Boston and one in Tubingen, decided to do
genetic screens with zebrafish and they thought that this would be a great vertebrate system to do large-scale genetic screens. And this was the first time this had
ever been done in a vertebrate organism, where they did an unbiased genetic
screen. So what do I mean by that? So what they did is they took a female, that is
just a normal wild-type female, and they took a male and they exposed him to a
chemical mutagen that actually induces lesions within the DNA that changes
actually how the information is processed within the DNA. They then
crossed this over several generations and ended up trying to identify clutches of
fish, or groups of eggs, that actually had a mutation of some particular phenotype
of interest. I have two of them highlighted here. The first one is this
one-eyed pinhead, and hopefully that you can appreciate, this is the showing from
the side of a lateral view of the zebrafish,
you can see the eye here. You can see the eye does not form very well in the wild-type.
Now if you look straight on, they’re looking right at me,
you can see the eyes are actually spread kind of very nicely apart, much like ours
are, but in one-eyed pinhead, hence the name, that they actually have cyclopia
and they form one single eye. Now, one thing that’s very important to note is
that the gene for that, that was a deletion that was induced within this
gene is actually cloned out and has homologues in humans and it actually
drives development of human embryos as well. And they get a cyclopia-like
phenotype if this is mutated in humans, providing a nice example that the genes
that we identify in zebrafish it can actually have implications for human
disease, and that is actually shown here as well in this bottom panel where,
hopefully you can appreciate, this orange dye that is staying right here with this
arrow pointing to it. This is the dye that’s added to the fish that actually
looks at hemoglobin levels within the fish. So in wild-type this is where the
heart is, sitting right underneath the eye here, and you can start to see that
this is where all the oxygen-rich blood is, right next to the heart. You can see
this is dramatically decreased in this mutant here and over time what they
found was that this actually provided a great model for anemic disorders in
humans and when they clone this gene out it was a [something] transporter gene. When that
is mutated in humans you get a very similar erythropoietic porphyria syndrome,
once again highlighting how important this, these genetic screens for, and so
for me, I think that several of these mutant phenotypes, because they resembled
genetic human diseases, provided wealth and knowledge. And just the sampling of
these phenotypes really kind of set the stage for why zebrafish could be used to
do these kinds of studies and actually what we would learn from them and what
this could contribute to human disease. Now what, for important, for our studies
and what I’m talking to you about today was cancer formation, and one other a
side note from this was that they actually found that these fish did
develop cancers in the laboratory in addition to in the wild, as I told you
before, and you can see here the fish, these are just papillomas,
these are carcinomas that form on the outside of the skin, and you can see that
those with a white bumps over the body and a blow-up of that is here. Now when
these little overgrowths form on the epithelia coating of your organs it’s
called a carcinoma and not a papilloma, and when we look at a
carcinoma from a human and a zebrafish they look morphologically similar. Actually, if I took the names off the bottom you’d probably not be able to tell
which one was the fish and which one was the human, suggesting that the things
that we find by studying zebrafish cancers would largely be applicable to
human studies. Now this was important for us and kind of set the stage for what we
want to do so when I came to Judy’s lab not very much had been done with the
actual epidermis of the zebrafish and so we set out to actually set up a model
where we could actually look at how these carcinomas form on the epithelia
that code our organs now we didn’t want to have to go in and look at all that
pithoi coding on mice or get this from human patients we wanted a system that
we could study it very rapidly like I had been telling you so we use the world
zebrafish epidermis and this is just a cartoon schematic of what it looks like
but if you kind of image right here on the side this is what you would see this
is the outer epidermis of the fish this is not like our skin that s Lux often is
it has a dead layer this is a living epidermis on the outside of the fish
here and they actually use this as though as their long early on in
development they don’t have gills at this stage so they’re actually oxygen
exchanges going on across the epidermis here now what I hope you can appreciate
is I put it right next to the mammary gland so this is a schematic of the
mouse mammary gland where they have luminal cells in my
delia and what you can hopefully appreciate is that they share a very
similar organizational similarity here where we have these bottom blue player
cells and this is a market called Carrick’s and it’s expressed in this
outer surface layer and then we have a marker of stem cells called p63 which is
these green dots and you can see that they’re very evenly spaced much like
they are and the mammillary going up athelia so we feel that this early
larval epidermis provided a great proxy to study the epithelia coding our organs
and provide a great way to study milk medically relevant epithelia in vivo
very rapidly now I want to get back to our model about what we thought and this
is that if we actually start to have these personal space issues within the
cells and this crowding that you can actually kick these cells out and this
is a process I told you we call it chronic induced extrusion and so I’m
gonna tell you a bit more about the extrusion process and how we think this
is regulated and what this actually means for cancer so this is just a
simple schematic of a dying cell with an epithelium so these outer cells at the
living once the yellow ones and the dying cell here in the middle is the
blue one and so what basically happens is when a cell dies it sends signals to
its neighbors that it needs to form a ring of actin and myosin around this
dead cell it’s going to help it squeeze that dying cell out so it starts to
contract it down over time and it pinches that cell and eventually it
leaves the tissue and it has been estimated that each of us lose a mass of
cells equal to our body weight over the course of the entire years who are
almost turning over all our feeling up within a year now not all epithelia turn
over at the exact same rate but I think this violates that this process has to
be very tightly regulated now this also illustrates that I told you that these
were dying cells now when I came to the web
most people had just hit these cells with chemicals and tried to figure out
what was going on with them we chose to actually just look and see how they
normally turned over and when we did that
so here’s one more example sorry about that with a this is actually just what
extrusion looks like within a cultured model just
letting you know that the cartoon was actually based on real data here and it
kind of looks like a flower pattern or rosette where you actually have these
are the live neighbors and they form this actin-myosin ring and have started
to squeeze out and this is the cell popping out of the layer now this once
again was a dying cell and you could tell by the nuclei being fragmented here
now is this really happening all over in our body I’ve only shown you a cartoon
in a tissue culture cell model but this does happen all we’re going to actually
use remove cells from many different type of epithelia and I’ve actually just
shown two examples here the first one is the bronchial epithelial layer your
lungs and you could see a cell popping off here
and the other one this best exemplifies this is the top of the intestinal villi
where there’s actually zones of extrusion at the top where you can see
these cells just pop off at a given frequency within the the tops of these
intestinal villi so when we’ve actually went back and started looking we
actually chose to look at the colon and see okay when these cells are being
eliminated from the tops of these zones of extrusion now they actually dying or
they just how are they turning over and when we did this what we actually found
was that the majority of cells were actually not done which is quite
surprising and we call this live cell extrusion so these cells have all the
hallmarks of extrusion but they do not appear to be done and that is actually
shown by this green stain here this is a marker of cell death so when cells
actually start to undergo the death process they turn on this marker and we
can stain for that and look for it and these cells and try to figure out if
they’re dying or not where we found that only 20% of these were dying at any
given point whereas 80% of them were still alive when they were being
eliminated from the tissue now this is the first time that anybody had ever
seen this everyone thought that the cell had to die to actually leave the tissue
now we’re changing the dogma here and actually showing that live cells can
actually just be kicked out and we wanted to know well why is this is this
a personal space issue now we found by looking at other systems we use the
developing zebrafish epidermis here like I just described you at those
larval stages and we found the exact same thing as we did in the human colon
samples where 88% of the cells that we found being kicked out of the tissue at
any given time were alive where is only 12% of them were dying now we also found
the same thing in tissue culture cells and where the percentages are relatively
the same where at every instance they were enriched for live cells being
kicked out from the tissue and dying cells just represented a small fraction
of what was going on so actually why would these cells being
kicked out so when we went back and looked we try to figure out right around
these cells was were there more nuclei around were there more cells in that
general area so what we did is that we actually measured the we counted the
number of cells either near these extrusion events or further down away
and infer this was for the colon for the fish we counted the cells right near the
extrusion event or further back and in culture we counted them right around
here or in a space that was near cell divisions and in every instance what we
found them was that there was more cells they were increased numbers of cells
right near where we found all these extrusion events this actually appear to
be 1.8 fold more crowded around every extrusion event that we witnessed within
these tissues this suggested that as these cells became more crowded they
kicked out a lot of cell so it’s kind of like an internal bouncer mechanism when
they got too many people they figured out who is the weakest link and they
sent them packing and sent them out the door and that is just shown here by a
little schematic where in the human colon these cells are actually born at
the bottom and migrate up and I told you that there’s these zones of extrusion at
the top now we think that this is happening on both sides of the colon so
there’s a force actually being applied and kicking these cells out when you
look at this in the zebrafish epidermis the same thing is happening although
it’s just tilted to the side mountains we’re looking at the fin where the cells
are born further back they actually start migrating and moving but they
converge at a common point where it creates some
or attention or a crowds own of crowd I mean we like to refer to it and it
actually pushes these cells out of the tissue and they have no choice but to
leave and this happens actually why they’re
still alive we found that this also happens in cell culture models so as I
told you this was had previously not been seen before and it was not known
that live cells could leave the tissue at all this has large implications for
cancer because of tumor cells could during metastasis I told you earlier
they actually leave the primary tumor site and go and leave and those cells
are alive long this is happening so it could be the tumor cells tumors actually
co-opted this mechanism to actually use for metastasis but what we really wanted
to show was what does this actually mean for the initial steps of cancer like
what I first talked to you about so what happens if we block this process now and
when we do that we went into the fish and we use some genetic tricks to be
able to do this this one in particular is just actually throwing on a chemical
compound that blocks a certain gene of interest that controls this process and
when we do this this is just the fin of the developing zebrafish here and this
is the spinal cord in the middle and out here is just the simple of the epidermis
that I showed you earlier and here is us standing for the nuclei within these
cells so you can just kind of appreciate at the ultrastructure level what is
going on now when we treat with this compound that blocks a lot of cell
extrusion now we start to see piles of cells forming on the sides of the fin
right where I told you that these zones of convergence or areas of high crowding
occurred so this is where we always saw these live cell extrusion events and now
when we block it we start to see cells pile up in those areas and that is shown
here as well you can see the nuclei are nice and evenly dispersed throughout the
fin and here they just form these very big clumps now this is more easily seen
in a transgenic fish with the skin outer
skin label and when we do the same treatments you can see either using this
chemical compound or inducing genetic perturbations that we it can actually
form little piles or aggregates of cells on the sides of the skin of the fish
these very closely resemble carcinomas that we’ve actually seen in human
patients so we feel that this provides a very good proxy for studying these
carcinomas in the fish model now I have been highlighting the whole time about
the visualization or the ability to look at what is going on here so we think
that this may represent initial step in cancer formation but we really want to
watch this process happen and since the fish will allow us to do that we want to
try to take use of these this transparent nature of the fish to watch
this form so this is where we turn to the fancy microscope compared to the one
I showed you earlier and then these two little technicians that help me out here
and so this is the outer this is the fin of the epidermis and what you’re going
to see here in just a second is an actual pile of these cells actually
forming at the top up here so we’ve added that compound that blocks
live-cell extrusion and now what you’re gonna see is the massive cells actually
forming in real time on the epidermis of this fish so we feel this is extremely
powerful because we start now we’re starting to actually be able to
visualize these very initial steps of what we think how cancers actually start
to form within these epithelial tissues so within these carcinoma formation we
think that we now have a nice way to start to be able to visualize these
processes so what does this mean for our model well I told you earlier that we
think that these cells are born at the bottom and start to migrate up and
they’re actually forced or get kicked or pushed out of the top of the colon so
what happens in cancer well we know that the cells to actually start piling up
here and we think this is the initial steps of cancer formation but I think
that what’s really important we still don’t understand
actually how the cells sense the force around them so they have to know that
their neighbors are somebody is gone awry how do you what actually drives us
and what actually causes these initial cells to start piling up here and so we
kind of need to tap our inner Yoda now and go back and start figuring out how
and why these cells are piling up but now you have a great way to start
watching this and we can start to use other chemical treatments to see if we
can alleviate this problem and so this leads me to the second question or
second part of the talk is can we use zebrafish to identify new therapeutic
treatments to prevent cancer formation or invasion so this has been done by
have multiple different groups and so this was not our idea but I thought I
just outlined the strategy for you can give you a few examples of actually this
types of studies actually going on here at the Huntsman Cancer Institute using
this strategy to try to identify new therapeutic compounds so basically what
they do is you cross these fish and I told you they give hundreds of eggs and
so you just put them in a dish and then you also take these chemical compounds
and here at the Huntsman Cancer Institute we have a library of these
compounds that are already been approved by the FDA but we just don’t know what
they do yet so people have started screening them that way if we find they
affect a specific prosthetic interest they can already start moving more
towards clinical trials within humans. So what they do is they take a unique
compound and put it in each of these wells and put a few eggs in each of the
wells as well. Then you go through under a fluorescent microscope and you can
just, or a basic dissecting microscope, and start screening the fish for the
phenotype of interest. In this particular case they wanted ones that turn red and
I’ll show you why this is important in a minute. And doing that, you know which
compound you put in that well so then if you can identify that fish you know
exactly what compound targeted that specific process of interest and this
allows us to actually identify new drugs that can be used to treat human disease. So this is where some of the researchers at HCI come in, and I’m gonna tell you a story today about Dr. Nick Trede and Dr. Jones who
worked together to identify a new compound to actually treat adult leukemias. And Dr. Cairns has also been instrumental in this process although I
don’t have time to talk about this work today. So there’s a zebrafish model that
Dr. Trede developed of T-ALL. This is t-cell acute lymphoblastic leukemia and
this represents about 25% of most leukemia cases and I should just start
by saying that there’s approximately 40,000 leukemia cases a year and the
surprising fact is that they have a very high death rate of approximately 50%. This t-cell T-ALL is the most common childhood cancer and I think even more
surprising the mortality of this one is 20% for children and 40% for adults
despite intensive research over a great deal of time. Now Dr. Trede a developed a
method here where he could actually label the thymus the t-cells are born
within these zebrafish and that is just shown as this green dot right here. Now these are the warble zebrafish but when you look in the adult that
green spot still stays there but if you can induce leukemia formation in these
fish now you can see that these cells actually take over the fish and they
become bright green and it’s very easy to actually just visualize and watch and
you can see these fish very easily underneath the microscope. So they use
this little genetic trick as a screening tool where they wanted to look for the
green spot within the fish, so this is the thymus, and they did what I told you
before where they put a compound a unique compound in each well and they
looked over time and what they wanted to find was ones that had no more green
spots so these were ones that eliminated the fluorescently labeled green t-cells
within the fish. So they screened twenty six thousand compounds and I just wanted
to throw that out there for the sheer scale of what they did is a tour de
force so to speak. And so they found that was a surprisingly large number of these
fish had no these compounds had no effect on the
zebrafish at all you can see that they just left a green spot where the thymus
was. Now, they found 2,000 or so that actually just killed the fish or made
them very sick so they didn’t want to study those but what was very nice
is that they found 21 compounds that had a very strong effect on t-cell
development, in particular the thymus. They had a few positive controls that
did the same thing but that’s provided in 21 candidate compounds that to
actually start targeting T-ALL. So this is the success story and that they
had one compound in particular called LDK where the vehicle is just whatever
the drug was diluted in so in this instance I think it was DMSO and so this
was just a the wild-type or the normal fish that gets leukemia and you can see
that the green spreads over time it becomes deceased by a day 40. Now when we
treat with this this new compound that they identified you can see that the
green cells within the Leukemia actually start to disappear over time and they
even become almost completely resolved by day 40. Now when you look at the survival
curve for that so what actually happens to these fish is this actually improving
their survival at all? It is. And so you can see with just the vehicle treated
alone where these fish are bright green most of them are dead by day 40 or
almost all them, whereas when you start to treat with this compound now
they have a 70 percent survival rate. So they went from actually screening a
compound in the fish to actually treating an adult fish with leukemia and
curing this fish and increasing survival rates which I find is pretty dramatic. Now they didn’t stop there they went back to our friend the mouse and did these
studies in mouse as well and what you can see here is just a picture of Mouse
and these very bright spots here are the tumor of a leukemia within the mouse and
the brighter red or yellow they are the more of tumor volume there is within
that mouse. So you can see the vehicle treated alone these are very bright spots and
has a normal tumor birth when they actually treat the mouse now with this
chemical compound it dramatically reduces the amount of tumor burden
within these mice and you can actually this is quantified here based on this
fluorescence where the blue blockers are actually this vehicle treated and the
purple bars are the LDK treated you can see that it dramatically decreases the
amount of tumor burden within these mice suggesting that this works in two
different in vivo models of leukemia and this provides a great hope considering I
told you the mortality rates of this cancer is so high and so I thought this
provided a great example of something that was actually done here with novel
compounds to identify a therapeutic treatment option for a leukemia. Now the second story I’m going to tell you
along the similar lines is the zebrafish model of skin cancer and the reason I
bring this up is that skin cancer is the most common common type of cancer and
there’s three basic types the squamous cell, basal cell and these
represents two different types of carcinomas, there’s approximately 3.5
million cases of these each year. Now probably more importantly than most of
the most of us are worried about that are exposed to the Sun are melanomas and of
these there’s 76,000 cases each year but the more important fact I think is
that of the of the 12,000 deaths that are associated with skin cancer 9,000
come from melanoma and so most of these are very aggressive and most of them are
very deadly and currently there’s very few effective treatment options. So once
again, there is a push to be able to identify a new therapeutic treatment
options to help prolong survival in melanoma patients. Now the Stewart lab
here at Huntsman Cancer Institute took a slightly different approach than the
Trede lab had taken before. Now that what they actually wanted to do was to
identify new targets within the melanoma that could actually be used the genes
that were either up or down regulated or changed their expression within these
melanomas that help drive this progression forward. They felt if they
identified these new targets then you could start identifying
therapeutic compounds that specifically target that gene of interest. And so what
they did is build on previous studies that had looked at expression levels in
human melanoma patients and what this study in particular found was a gene
called Axl, Axl, was significantly up-regulated in aggressive melanoma samples
and so this is one this is just a simple survival curve of patients and they took
a sample of their tumor over time and they saw the ones with weak Axl
expression did much better than the ones that had strong Axl expression within
these tumors over time. And they also found that the primary melanoma tumors
very little of those tumors expressed Axl whereas the ones that actually
metastasize, and I told you earlier that the metastasis is the more deadly
portion of the disease, they found that this expressed Axl very highly.
So the Stewart lab felt this is a great target to try to figure out how this
occurs in melanoma. So what they did is they use this fish model that lacks
pigment completely, they they add a system that can read it rescue the
pigment while also over expressing this gene Axl, but they’ve
tagged it with a green fluorescent so they can watch it over time.
Now when they do this the fish develops a massive melanoma
on its back and now you can see that in addition to this one there’s other areas
of the fish that did not look good but I totally fluorescently tagged it and you
can see that the tumor is actually green suggesting that Axl over-expression actually can drive melanoma formation within the zebrafish. And when
they did this, when they looked at these transgenic lines they found that when
they did this treatment over 50 percent of these fish form tumors whereas the
control fish only about 13 percent actually form tumors over time. Now this
is actually identifying a gene that is associated with melanoma progression.
Oops. And they actually just measure the tumor growth
and invasion of these particular tumours and those are ongoing now. But what they
actually wanted to do is they teamed up with a group of chemists here at the
University of Utah and at Huntsman Cancer Institute in particular and they
actually designed a specific inhibitor that would target this Axl kinase. And
so this is just a little schematic or a model showing this is the drug sitting
in here and it’s kind of sitting in this binding pocket of this protein of
interest so what they wanted to do was identify an inhibitor so this is
something that would block the function of this particular gene of interest, this
Axl kinase. So they designed this inhibitor and they think that it’ll go
in and sit right within the way this protein is folded and prevent it from
functioning normally within these cells and they had very good of prelininary
results that show that treatment of invasive cancer cells and culture that
inhibited this behavior and that was published a few years ago. So they moved
the Stewart moved on with this and they actually started to treat larvae
with this and I found this result very surprising when you look at normal fish
I think these are at 48 hours on these larval stages that you get these I
showed you earlier these pigment cells on the back of the head so these will
form the stripes in the adult fish but they kind of spread out over the back of
the head over time and they form this very nice gap and you can see that they
kind of form little projections over time. Now in the drug treatment guys you
see they actually just form a big black spot and it turns out that over time
treatment with these with this drug prevents the melanocytes from actually
migrating away during development which is actually quite surprising . So if
you’re talking about wanting to identify a compound that prevents metastasis of
melanoma cells preventing the migration of melanocytes is a great way to start
and so they thought this is a very promising result and that it was worth
following up on that they actually could prevent the migration of melanocytes. So now what they hope to do is take this melanoma model that I told you
about earlier where that they overexpress this Axl and they can drive
melanoma within these fish. So what happens now if we treat with this
compound can you actually prevent the fish from actually forming a melanoma? If
that is the case then that would provide a very nice therapeutic treatment option
to prevent melanoma formation or at least the most aggressive types. And so I
thought this was a very nice example and all this work I have to say was very
nice of Laura Jimenez and Rodney Stewart for sharing because none of this is
published so far yet. And so in summary, what I told you today is a little bit
about just kind of how cancer is caused by cells that go wild and initially we
just kind of started out with this basic schematic of how cancer forms but
hopefully I’ve convinced you that we’ve at least been able to start to watch some
of these early initial steps. Now I also think that it’s it’s highlighted from
our studies that it’s important to know who your neighbors are or more importantly
if they know who they are how this works it seems that many of these cells kind
of forget their past behaviors and this is what initial leads to this initial
piling up. Now hopefully I’ve convinced you the zebrafish provide a unique model
system to visualize these initial steps of cancer formation we can actually
watch this under the microscope and take movies of this which has been extremely
informative instead of just taking single snapshots. And finally, this last
part, and this was actually used from the American Heart Association because I
told you they can regenerate their heart so people are also using these drug
screens for heart regeneration compounds but I think that they can provide some
hope that we can actually use zebrafish to identify new therapeutic treatment
options for aggressive human cancers and so I’ve just given you two examples of
what’s actually happening here at the Huntsman Cancer Institute on this front
but this is happening all over the country at other Institute’s as well and so
I think that the future’s bright for trying to identify new compounds to help
treat human diseases. Well thank you for your attention.

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