Hi. My name is Hans Clevers.
I'm a scientist from the Netherlands.
I'm giving three lectures for iBiology.
This is the third one,
and in this one, I will describe
how we are employing organoid technology
-- starting from adult tissues, not from iPS cells --
for disease modeling and ultimately for personalized medicine.
And the first example I will give you is cystic fibrosis,
which is currently the best developed.
To summarize what I've said in the previous two lectures,
it is possible to exploit
the repair capacity of tissues
by taking a small sample from that tissue
-- it could be a mouse, it could be a human,
healthy or a diseased human, a diseased tissue --
put it in culture, in 3D,
in a defined growth factor cocktail
that differs subtly from tissue to tissue, to be optimal,
and then you have these cells growing.
I've shown in a previous talk
that you can use these cells to repair tissues --
you can actually transplant them.
So far, this has been done in mice,
and my colleague in Japan,
my collaborate Mamoru Watanabe,
is gearing up to do the first in-man trial
for gut organoids in inflammatory bowel disease.
We realized this is one application of the technology.
But a much more rapid application
would be the use of these organoids
for personalized medicine and for disease modeling
outside of the individual from which you got the tissue.
As you can see here,
it is possible with the adult stem cell-based organoid technology
to grow a large number of different tissues.
Of note, these are usually the epithelial elements of those tissues,
so they are the sources of carcinomas.
They're also the business ends of many, many organs
like the lungs or the pancreas or the liver or the gut.
But these organoids will not have nerves;
they will not have blood vessels;
they will not have immune cells;
they will not have the microbiome,
unless you add them to a growing organoid.
What is known now, and observed by many labs,
is that if you do so,
the additional components will find the right location
and will build more complicated structures.
They're starting to look more and more like the real organ.
But again, our version of organoid technology
produces the epithelial part of... of organs.
Cystic fibrosis is a disease...
a very common... the most common hereditary disease in Caucasians.
It affects a single gene that encodes a chloride channel,
and that chloride channel's function
is very important to keep mucus layers moist
and to keep mucus moving through gut or liver
or, for cystic fibrosis,
very important for the lung.
If you lack the channel,
you cannot transport water to the mucus layers.
The mucus gets very sticky --
you see that happening here.
Bacteria get trapped in this mucus.
They will proliferate.
You get chronic inflammation.
And cystic fibrosis patients get chronic problems --
deteriorating organ function, particularly in the lung.
And nowadays, without treatment,
a patient is not expected to live beyond 35 to 40 years.
Vertex, a company in the U.S.,
has come up with an incredible molecule,
or set of molecules,
that correct the function of this gene.
And they work in a very unexpected way.
What they do... so, they...
so, typically, CF patients can produce the protein,
but the protein has a mutant amino
or it lacks one amino acid.
As a consequence, it doesn't fold correctly,
and then it gets degraded inside the cell.
Or if it does fold to some extent
and gets to the surface,
it doesn't gate properly,
and then, again, it doesn't function the way it should,
and the patient is in trouble.
Now, what Vertex has developed is a set of currently two molecules
that can correct either the folding
or the gating of a mutant protein.
And as you can understand,
this will be a very mutation-specific drug,
because if your problem is here in the channel
and the drug acts here,
it would not correct the problem here.
It will only correct problems at the site where it binds.
So, what Vertex did is they focused on the largest group
of cystic fibrosis patients.
You can see here the gene.
There's a large number of different mutations,
but there is... about half of the patients worldwide
carry the same mutation,
a deletion at position 508, a phenylalanine.
This misfold...
it leads to misfolding and gating problems in the protein.
And you see this in the green area, here.
These are all the patients
with that particular problem.
So, in principle, the Vertex drug will cure these people,
but it will not help all of these other patients
that have rare mutations.
So, why is it that CF is so common?
And it's generally believed that this has to do
with the fact that in medieval times,
particularly in Europe,
cholera was occasionally epidemic.
The toxin that is produced by the cholera bacterium
acts directly on the cystic fibrosis channel.
It opens it up.
And if you happen to be a normal individual
with normal CFTR channels,
and you have this bacterium in your gut,
you produce a massive amount of diarrhea.
The channel open; liquid goes out massively.
It's not dosed in any way,
and the patient will produce up to 20 liters of diarrhea,
will dehydrate, and will die very rapidly.
If you happen to be a carrier or if you happen to be,
even better in this situation, a CF patient,
you don't produce this massive amount of diarrhea.
You produce much less as a carrier
and probably no diarrhea as a CF patient,
and you will survive.
And for that reason,
mutations in this gene have been accumulating in recent centuries in Europe,
and it's apparently most common
in Caucasians in Europe and in the U.S.
These mutations have occurred many, many times,
and that's why there are so many different ones.
You have to realize some of these mutations
are unique to a single family, or a small village,
or a small group of people who have then
scattered out around the world.
So, first of all, business-wise,
it is very difficult to find a commercial plant
that will allow you to develop a drug
for that particular group,
because it's so few patients.
Also, it would be impossible to ever collect enough patients
to do a statistically relevant trial on these patients
to show that it works.
So, essentially, these people were left with no treatment.
These people were helped by the Vertex drug that is...
its trade name is Orkambi.
What we realized...
this was done together with the pediatric...
pediatric hospital in Utrecht with Kors van der Ent and Jeff Beekman...
Florijn Dekkers was the first author on the first paper, here...
was that you can mimic the massive diarrhea
that's caused by cholera toxin in mini-guts.
On the left, here, you see the mini-guts
of a normal individual, stained in green.
The movie is run over one hour.
They're looping.
We add cholera toxin
or we add a chemical called forskolin
that has the same effect.
What happens is this immediately opens the channel,
and the mini-gut will swell,
because now water is pumped into the lumen of the mini-gut,
as if you're producing cholera diarrhea.
There's no exit to these mini-guts,
so they have to swell.
And you can see they swell about 2-3-fold
over about an hour.
In the middle, here, you see the mini-guts.
And this... these are... I should stress, maybe,
grown from a rectal biopsy
. So, this is painless.
Little kids really don't care very much about a rectal biopsy.
It takes about a week or two weeks to grow them up
to have enough for this assay.
This is... what turns out to be a CF patient
with a delta 508 homozygous mutation,
so a patient that is eligible for Orkambi.
You can see that they've grown very well
-- there's enough of these mini-guts --
but they almost have no lumen.
And then we add forskolin.
When we open the channel the way cholera toxin would do this,
essentially nothing happens.
This is why this patient
would survive a cholera infection,
but also the reason why this patient has cystic fibrosis
and all the problems in, in this case,
his organs.
If we now take mini-guts from this patient
and we preincubate them with the Orkambi drug,
we now all of a sudden realize
that we can restore, functionally...
CFTR channel function.
As you can see, the swelling, here,
over about an hour
is the same as what you would see in a healthy control.
So, these are cystic fibrosis mini-gut organoids
treated with Orkambi,
and this is a sign that, very likely for this patient,
Orkambi will work.
Now, based on this assay,
Kors van der Ent, the treating physician,
a professor in Utrecht...
was talking to Fabio.
And Fabio was very sick at the time.
Fabio is a CF patient,
but he has an unusual mutation
that so far has only been seen in his family.
Originally, we knew about him and an aunt.
There turns out to be a third family member, I believe.
The mutation was never seen anywhere else in the world,
but it was pretty close
to where the delta 508 mutation was located,
so it was assumed that possibly
he could respond to this drug.
The problem is the drug, at that time, was not registered.
Also, it was a very expensive drug.
Insurance companies would not just give this drug to us.
You also have to treat a patient for about a year to decide if it...
that it doesn't work.
And there's nothing else,
so patients like to have this drug,
because it's their only hope for a...
for a cure, at least... or for some help.
So, Florijn Dekkers made mini-guts from a...
from a biopsy from Fabio and tested Kalydeco --
this is essentially one of the two components.
And he responded dramatically well to the...
to the treatment of his organoids with Kalydeco.
So we... so we used, essentially, his mini-guts as an avatar of Fabio.
This was, then, enough reason for the hospital to say,
okay, we'll make the drug available,
and within the matter of a week
it was clear that he responded dramatically well.
As I said, he was very sick.
But after this, he's actually now back on the sports field.
He plays field hockey.
He's a very good field hockey player.
And it looks like he's now cured of his disease.
He would never have gotten this drug
if the organoid test would have not existed.
Now, based on his case...
so, De Wereld Draait Door is the most popular Dutch talk show.
He told his story here,
with Kors, you know, about what happened.
This then made the Ministry of Health in Holland
decide that Orkambi should be registered in Holland...
these Vertex drugs.
And now the registration of Orkambi
says it can be given to every delta 508 mutant,
the common mutation,
but it can also be given and reimbursed
to every patient with these unusual mutations
with a positive organoid test.
So, this is the first time, we think,
that organoid technology has really entered regular health care.
This is Holland.
We're currently looking at...
to build a large biobank of all the CF patients...
all the rare CF patients in the entirety of Europe.
In Holland, we have 1500 patients.
Our biobank currently holds about 700-800,
and we hope to get all of them
and will expand this in Holland.
And this can actually be done in a simple...
in a single laboratory center,
where the organoids are also stored.
So, that is one example
of how organoids can be used in a personalized medicine setting.
A much larger clinical field,
but also a richer field, would be cancer,
and I'll give you some examples of how we have
developed organoid technology
to get insights into cancer processes,
but eventually to also maybe design assays
that are very similar to the CF assay,
that would allow us to decide
whether a drug will or will not work
for a given patient.
So, one thing we've been very active with
-- and now there's multiple other labs in the world that do this --
is to build large biobanks, realizing that...
that cancer, although it can be,
you know, colon cancer,
every case is essentially unique.
And this is actually in Dutch.
Gezond means healthy; Ziek means sick.
So, what you saw is we can grow
the healthy tissue of a cancer patient,
and we can also grow the tumor tissue
from the same patient.
Grow them side by side.
And you can see, here,
the number of carcinomas for which we have built large biobanks,
in some cases of hundreds of patients.
We can sequence -- DNA, RNA.
That could be done directly on the tumor.
But we can also functionally test,
like we did for CF or like you would do for a bacterium
with antibiotic testing...
we can test resistance or sensitivity
to a large set of clinically used cancer drugs,
and also combinations of cancer drugs.
This works very well, at least the testing.
We are currently embarking,
or have embarked, on trials,
where we cannot instruct the oncology...
the oncologist to change treatment of patients,
but we can actually follow patients in time,
see what the treatment was,
and actually see if our organoid testing
had predicted the outcome.
So, did we predict correctly
that a drug would be working or would be not working for a patient.
You have to realize that generally, overall,
patients that get treated with anticancer drugs...
about 40% of them will respond to the drug.
About 60% will not respond.
100% will develop cost,
but more importantly will develop all the side effects.
And they'll... they'll then experience
a delay in treatment with a drug that does help the patient.
So, there is a very, very strong incentive
to find... you know,
when you start treating the patient, find the best drug,
not by statistics, not that you're in a group
where we know 60% will respond.
You would like to know 100%
that that patient will respond to the drug.
Now, we think that the organoid technology holds this promise.
We were scooped, as happens occasionally in science,
about a year ago by a paper by Vlachogiannis et al in Science
that showed that this is indeed the case.
You can grow...
and this is the procedure...
you can grow organoids, and they followed a number of Phase I and II trials,
and they showed an extremely high predictive value
-- up to 90-95% --
of predicting whether a given tumor
will or will not respond
to a given combination of drugs.
And there's now multiple centers in the world
-- and we are one of them --
that are trying to establish this as a more regular and, possibly,
like I just told you for CF,
incorporate this into the normal clinical care
that in... in cases where an oncologist
is not really certain what should be given,
you could actually use organoid testing...
tumoroids, we call them...
tumor organoid testing to find the best drug combination.
This is what they look like:
normal tissue; tumor tissue from one patient;
another one... normal tissue from the second patient.
So, they don't really look very different.
They can be screened very well.
Without going into much detail,
here we have about five or six colon cancer patients.
These have an APC mutation.
They are... they will not respond to Wnt blockers.
This one would... is an RNF43 mutation...
you don't really need to know the details,
but this one would be predicted to respond to a Wnt inhibitor,
in this case a porcupine inhibitor.
And in a blind screen,
you can see how well this one patient
separates from these other patients.
So, we really believe there is a lot of evidence, now,
that cancer organoids can be used in a predictive fashion
in the clinic.
We made... in the course of these studies,
unusual observations.
The first one, and I will not illustrate with this slide,
but we find that almost all cancer drugs
are better at killing normal cells
than killing the cancer cells of the patients.
You would hope that cancer cell drugs
would kill cancer cells, but they don't.
There are very few that are specific.
You have to also realize there are no normal cell lines of normal...
that represent real normal cells.
And we think that organoid technology
now, for the first time,
allows drug companies to screen their drugs
against healthy cells and cancer cells,
and possibly improve on the therapeutic window
by developing drugs that will be specific to cancer cells,
rather than be specific for cells that divide,
or something like that.
So, that's the first somewhat unexpected finding.
The second was really counterintuitive.
What you saw in this movie...
these are normal human colon organoids,
and there were several mitoses
that proceeded rapidly,
and in these normal colon organoids,
they always result in two healthy daughter cells.
It turns out that normal cells are much better at dividing
and at expanding in organoid culture
than cancer cells.
Very counterintuitive.
But to show you what happens in these cancer cells,
here you see two nuclei.
It turns out to be a single cell with two nuclei.
This is not normal in the colon.
When the chromosomes condense,
there are up to 100 chromosomes
-- you realize... this is also not normal.
When the cell, then, tries to segregate these chromosomes,
it has a very, very hard time doing so.
Here's another cell that originally generates three nuclei,
tries to resolve to two nuclei.
Here, essentially, the cells gave up
and they just died.
And this is what we typically see.
So, the normal tissue,
under these growth conditions,
grows exponentially, and very fast.
And the fastest-growing tumors
will actually have about that speed,
but almost all other tumors
will grow slower than their normal counterparts.
So, how do we interpret this?
What we think is that
normal tissue is very well designed to proliferate,
but it's also very well controlled.
So, it will proliferate as fast as it can
to repair a defect or a loss of tissue,
but the moment that...
actually, that the cells that are rep...
that have died are replaced,
the system settles back into a resting state.
We can do that in culture.
We can take the growth factors out,
and the organoids will basically just sit there,
and they will not grow.
The cancer cells are like zombies.
So, they cannot sprint.
They are stumbling around, pieces fall off.
But the big difference with a normal organoid is they will never stop.
So, eventually they will... they will get the patient.
And that is their problem.
Meanwhile, you can see how much the damage is
that they incur.
So, they're pretty lousy proliferators, as cells.
But in the end, they will kill the patient.
This is a practical problem,
because if you get a tissue that's partly normal cells
and partly cancer cells
-- for instance, prostate samples always are mixed cancer and normal cells --
and you put it in culture,
if you don't somehow block the proliferation of the normal cells,
they will immediately take over the culture.
And after two or three passages,
you're essentially studying normal cells.
Now, that was not the intention of isolating cells from the tumor.
So, for some tissues, we have solved this,
and I'll give you examples of that.
But particularly for the prostate,
we don't know yet how to get
a 100% pure prostate cancer cell sample,
or get conditions where the normal cells
will not survive but the cancer cells will grow up.
A second application of organoid technology in cancer research
is the following.
And I think this experiment by Jarno Drost,
but it was also independently done by Toshi Sato,
who is now... who has his own lab in Tokyo.
And both of us realized independently
that the mutations you see typically in colon cancer
-- APC, KRAS, SMAD4, P53...
they really reflect the growth factor cocktails
that we have empirically defined.
And to illustrate this,
APC is a negative regulator of Wnt signals,
so we need to provide Wnt.
I'll show you, if we take it out of the medium,
normal cells will die.
But when cells are APC mutant,
they are predicted to no longer need Wnt
in their growth factor cocktail.
KRAS, a very common mutation in colon cancer,
is in the EGF pathway.
So, an oncogenic mutation in KRAS
would probably take away the need for EGF in the cocktail.
SMAD4 is a BMP inhibitor...
sorry, it's a transcription factor in the BMP pathway.
Noggin is a BMP inhibitor.
Empirically, we knew it was important.
We never knew why.
But cancer does the same thing
as we do in our culture system.
It gets rid of the BMP signal not by adding Noggin,
but by mutating the transcription factor that mediates the signals.
And then P53 we have added in this experiment
because it's mutant in the overwhelming majority of solid cancers.
So, what did we do,
and what did Toshi do independently?
Here, you see human, normal colon organoids.
If you would transplant these back into a mouse,
they would make normal gut epithelium
and behave well.
If you take Wnt out,
you can see they immediately die.
This is... this happens in a matter of days.
There's no viable organoid left here.
But if we at the same time
target the APC gene with four different guide RNAs
with CRISPR...
some are better than others,
but we get these large organoids growing
in the absence of Wnt,
so they only have EGF and Noggin.
And when you take one of these and you sequence them,
they turn out to be clonal.
So, there was a single cell
where we hit APC on both alleles,
caused two frameshifts.
APC is no longer there.
The Wnt pathway is heavily activated.
And it no longer needs triggering from the outside.
So, they only need EGF and Noggin.
The second mutation, KRAS, here.
This is an oncogene,
so we cannot just delete part of the gene;
we have to very specifically mutate a base.
We do this, again, with CRISPR,
and you can see that happening here.
CRISPR-CAS9 lands near the site that we want to mutate,
causes a double-strand break.
We provide a large, 100-base oligonucleotide.
This allows the cell to correct the break, here,
but at the same time, it puts in a mutant base.
Now we're no longer encoded at position 12
for glycine but for an aspartic acid,
which you see here.
Normal colon epithelial organoids,
be it APC wild type or mutant,
will die without EGF,
but when we have activated KRAS with the mutation,
they happily live.
They only need Noggin.
P53 is very easy to mutate with CRISPR-CAS9.
You can select the mutant cells
by adding a small molecule called Nutlin.
Nutlin interferes with the MDM2-P53 interaction.
It will kill wild type P53 cells very efficiently,
as you can see here.
But if we target P53
-- again, with four different guide RNAs --
we get clonal organoids.
And as you can see here, they have lost P53.
So, now they have three mutations.
They're very close to sort of the prototype colon cancer,
and they only need Noggin in the medium --
nothing else. No serum, nothing else.
So, for Noggin removal...
when we do that, we kill the organoids,
even though they already have three mutations.
But when we target SMAD4,
which mediates BMP signals,
we no longer need Noggin.
And now we have four mutations in.
And now these cells will grow...
not in water, but in medium
without any addition of any growth factors or serum.
When we now start transplanting
all of these different combinations,
only the ones with the four mutations, here,
will start creating tumors.
So, these are human colon...
synthetic colon cancer organoids
transplanted orthotopically into the colons of mice.
And only when you have four mutations
will they grow invasively,
and they will metastasize,
particularly to the liver.
And our pathologist tells us
they really look like the real thing,
like real human colon cancer.
So, this... I think this experiment illustrates two things.
First of all, it is quite feasible
-- and I'll give another example after this --
to create very well-defined human cancer models
in organoids.
You can do all sort of interesting things with them,
but if you want to convince a pathologist
that it actually is a tumor cell,
you can quite easily transplant them to mice
and recreate the morphology of a... of a real tumor.
And for this particular experiment,
what I found very interesting
is the fact that this growth factor cocktail
that Toshi Sato had put together totally empirically...
that, actually, cancers do the exact same thing.
They activate Wnt.
They activate EGF receptor signaling through KRAS.
They block BMP signaling.
And that's exactly what we do in our culture conditions,
emphasizing that cancers
exploit the normal mechanisms that stem cells use
in their normal behavior, in normal tissues.
Yeah.
And what I should probably also emphasize...
when we try to design culture conditions for a new tissue
-- like, recently, we did ovarian epithelium and ovarian cancer --
we look at what we know generally
-- we need to activate Wnt; we need to activate a tyrosine kinase receptor;
we need to block TGF-beta --
but then we look in the cancers of that tissue...
what pathways are blocked?
What pathways are activated?
And that usually is a strong indication
that we should really add or remove or block
that particular signal,
on top of the ones that are the basic organoid growth factor conditions.
So, a final story, here.
Benedetta Artegiani
tried to model cholangiocarcinoma.
This is a cancer in the liver of the bile duct,
of the cholangiocytes in the bile duct.
And particularly...
so, these are the cholangiocyte carcinomas already...
cholangiocyte organoids.
I already discussed them earlier.
I'm particularly focusing on the gene, BAP1,
that is mutated in a number of different tissues
that you see here.
It is known to be a tumor suppressor.
You need to lose function of both alleles.
In the fly, there appears to be a homologue
that's called Calypso.
And Calypso is very well-known...
very well-studied
and is really accepted to be
a member of the Polycomb repressor complex
that essentially takes ubiquitins off H2A
at position lysine-119.
There are a large number of papers
on how BAP1 causes human cancer.
Almost every paper has a different mechanism,
but none of those papers
refers to the role of what we think
is the orthologue of this particular gene in flies.
Also, the mouse models for BAP1
don't produce the tumors
that you'd see in humans,
so... so clearly the mouse is not a good model
to study how BAP1 causes cancer.
And Benedetta wanted to explore...
you see her last name, here,
on the paper that she recently published...
wanted to explore organoids
to see if she could learn how BAP1 transforms cells.
Now, she... she hit the jackpot right away.
Here you see a cholangiocyte organoid
from a normal human liver.
She's done this from multiple donors.
Highly polarized.
So, this is where the lumen is;
this is the apical side; this is the basal side.
Highly polarized, well-formed junctions...
this is a real epithelium.
This is the epithelium that lines the bile ducts.
The BAP1 organoids are dense,
have no lumen.
There's also no junctions.
There's no polarity.
So, they really lose epithelial characteristics.
And this would be... people would refer to this as EMT.
So, EMT is often defined by a few markers.
This is sort of functional EMT.
These cells... you take out a single gene,
and they lose any aspect
that would make them look epithelial.
So, BAP1 somehow,
we think, controls epithelial contacts.
And you can see that here, as well.
ZO-1 is a junction marker. It sits at the apex of cells.
You can see this, here, on the confocal.
Very nicely... the cells are high polarized.
Very close to the lumenal junctions,
you can see where ZO-1 is located.
The moment you knock out BAP1,
essentially ZO-1 is all over the place,
and you no longer see any functional junctions formed.
So, BAP1, again,
somehow controls the function of junctions.
When we make movies...
here you see normal cholangiocyte organoids growing --
thin-walled, highly polarized epithelium.
You can see they're quite dynamic.
You just knock out BAP1,
and you get these slimy monsters
that crawl around and that basically eat up
other organoids, fuse and merge.
There's no lumen.
There's nothing that makes them look epithelial.
These really are mesenchymal cells.
Yeah.
Now, most of the papers on BAP1 tumor suppressor function
claim a role for BAP1 in the cytoplasm.
So, Benedetta expressed BAP1 in these mutant BAP1 organoids
in a cytoplasmic version.
The greens that the expression works,
but you basically see no change
in the behavior of these cells.
But when she expressed it in the nucleus of these cells,
you can see that in a matter of hours
-- the whole movie runs over 24 hours --
she reestablishes a very nicely polarized
epithelial structure,
implying that BAP1 functions in the nucleus,
and not in the cytoplasm,
to exert this epithelial organization, in fact.
Lots of omics experiments
showed that BAP1 indeed is a chromatin modifier.
It indeed is the orthologue of the fly.
It controls the ubiquitination of this histone.
And here, for instance, by ATAC sequencing,
Claudin-1 is a very crucial
for epithelial behavior in cells.
The enhancers and the promoters
are totally closed in the mutant.
But the moment you reexpress
or the moment that you look in the wild type cell,
you see that these regulatory elements of this gene
are fully open, as demonstrated by ATAC sequencing,
implying that, indeed,
BAP1 is a regulator of gene expression.
And when we look at the genes that are controlled,
surprisingly... a small repertoire of genes
-- about 300 --
and almost all of the genes you can recognize
have a role in epithelial characteristics,
be it polarity or be it the formation of junctions,
etc, etc.
So, BAP1 is a chromatin modifier,
an epigenetic regulator,
that has the unique role, at least in liver cells,
to tell cells to be epithelial.
And if you lose it, you're no longer epithelial.
So, how does this play out for cancer.
If we transplant these cells
that look very ugly in culture into a mouse,
they do very little.
They make a little bit of an adenoma,
but then they get stuck.
So, they are not malignantly transformed,
although in vitro they look horrible.
What Benedetta then did is
she actually rapidly mutated four common genes
that are often seen in cholangiocarcinoma:
P53, SMAD, NF1, and PTEN.
These cells have both of these...
both alleles of each of these genes mutated.
They actually look pretty normal as organoids.
And when you transplant them,
they make... so, these are the organoids...
sorry... and when you do the same thing,
but on top you add the BAP1, you, again,
immediately see this strange epithelial to mesenchymal transition.
No longer a large lumen,
no longer any polarity,
no longer any junctional function.
So, as I said, just transplanting BAP1 mutant organoids
doesn't give you a tumor.
She then transplanted the organoids
with the four mutations that were wild type for BAP1
and the ones that,
on top of the four mutations,
had this BAP1 mutation.
Again, the ones with the four mutations,
you would predict to be extremely malignant,
having lost these four tumor suppressors.
They grow in a very benign way.
Our pathologist called them an adenoma,
definitely not a cholangiocarcinoma.
But the moment we now add in this fifth gene, BAP1,
we get this here.
We get a large stromal reaction.
We get an extremely malignant-looking epithelial component, here.
This is... our pathologist
tells us this is a real human cholangiocarcinoma --
recreated starting from a normal liver cell
by adding five different mutations
and then transplanting into a mouse.
So, we think that this technology
can be used as an alternative to genetically engineered mouse modeling,
for instance,
to rapidly assemble lots of mutations
and then make isogenic series of organoids
that have different combinations
but all started from the same cell,
and then test them...
either in vitro or test them by transplantation.
And with that, I'd like to thank you very much for your attention.
