So I sort of have a little.
So I'm delighted to have a guest speaker today, Simon Bouzouki, who many of you will know he is a colorectal surgeon.
Quite a successful academic at Cambridge.
I've come across him through a Cancer Research UK.
He's a very good work, which, uh, made him win two awards from Cancer Research UK,
one initial clinician scientist and then the second more recent and advanced clinician scientist.
I won't steal his thunder, but he's got a very exciting research programme, clinical practise focussed on laparoscopic and colorectal procedures.
And he was telling me yesterday that he also leads something which is new and important from the US,
called out the digestive cancer programme and bowel well.
So maybe he can tell us more about that. Um, he's he's lab programme.
He's mainly about the um, the whole sub clonal interactions play on colorectal cancer cell identity and behaviour.
Um, and we'll hear more about it. So I won't dwell on that.
But a very warm welcome. Uh, Simon, and uh, please thank you very much, Prof.
Thank you very much for the very kind invitation to come and speak.
I I'm talking on net, which really are not the main focus of my clinical practise or my scientific focus,
but I thought they were interesting because they're some.
They're interesting from a surgical perspective, and there's a lot of very interesting science around net biology,
which is being very, very poorly understood and poorly explored.
So I thought I'd initially just give a little bit of a background to on my
clinical practise in the scientific practise and the set up we have in Cambridge,
just to put everything into somewhat of a perspective. So our unit is similarly sized to the unit.
Here in Oxford, we have six contractual consultants who are doing purely colorectal colorectal surgery.
We also have three emergency general surgeons who are also fully embedded into our department.
So a quarter of their practise is EEGs work, and three quarters of it is colorectal surgery, including resection or what.
So we fully embed them and then we have one full time academic myself.
The department has a full spectrum of subspecialty practise.
There's a pelvic exam, iterative service, which many of you in the field when I was led by Nicky Fern had supported by James Wheeler,
who were both Oxford trainees originally, as well as John Moores and Justin Davis.
Our pelvic floor services run by John Morton and Michael Powell.
Early Rectal Cancer with Thames is run by James Wheeler and Nigel Hall, who have an anal cancer service,
a newly developed endometriosis service together with our gynaecology colleagues and
also the net service and the net service is really led solely by myself at the moment.
We have a new colleague who will be starting next month, who will be joining me with Nat Resections,
which will help because there's just not enough capacity for myself to do all of the net actions, as well as the standard cancer section of work.
So similarly focussed, I guess, to what you have to wear based upon the Cambridge biomedical campus.
And I don't know whether many of you been up to Adam Brookes recently, but it is expanding at an ever increasing rate.
The biomedical campus is now the largest by health related biomedical campus in Europe and has a very strong cancer theme to it.
Some of the new developments it is the hospital is based over here and so extends off the right hand side of this photo.
There's a lot of new scientific developments and industry on site,
so this building in the shape of a chromosome is the new laboratory of Molecular Biology, which opened about four or five years ago.
This is AstraZeneca as worldwide headquarters for research and development, and this is Royal Papworth, which finally,
after what seemed like an eternity, has finally moved from that village site in Papworth to being on campus with us.
This is the senior UK Cambridge test tube, which is where most of my research has been done to date,
and I'm in the process of moving into this building, which is the STEM Cell Institute, which is being relocated from being in town up here on site.
There's also the MRC Hutchinson Cancer Cell Unit and the Cambridge Institute for Medical Research.
There's going to be new children's hospital developed on this site over this site, which just got government support,
and we're in the process of applying on this site to develop a cancer research hospital.
But that will not have any surgery. Than it so you can.
So there's clearly a very strong co-location of clinical and scientific disciplines on campus,
which very much helps in interdisciplinary approaches to research.
We have strong funding from the Biopsy and Cancer Research UK and we're seeing UK major centre and and as I say,
with aims at being on site and MedImmune as well as outcome, we're having an increasing amounts of collaborations with industry.
The cancer focus is interesting and worth mentioning,
and that is there's not a strong GI focus on sight and this is historical based on the peers who are present there,
and it's predominantly breast, ovarian and early detection. So my science so I said my focus is mainly on colorectal cancer biology,
and it's about about understanding the interlinked behaviour between cellular identity and
behaviour in cancer and understanding why cells transition from one state to another.
And we're particularly interested in cells have received little focus to date differentiated cancer cells,
but we've identified that subpopulations of these can acquire up to the capacity or so interested in tumour evolution.
But today we're going to be talking about small intestinal net biology, which I became interested in when I started to do net resections clinically.
The techniques we use in the lab are very much based on using primary tissue,
and that's why it's key to have your science based on a closed location to where you're carrying out your clinical practise.
We do a lot of organoid biology using CRISPR targeting on normal organoids.
Singles out work so single cell RNA seq. We use a technique called lineage tracing, which I'll come on to shortly,
and I'm increasingly interested in using game theory to understand clonal interactions.
So nets to give you some background to them.
They they are very rare.
They used to be called carcinoid, however, of about sort of 10 15 years ago, the nomenclature changed to neuroendocrine tumours.
And these are this is this sort of figure on the left shows the common sites below the diaphragm to find that the three common places really are long,
small intestine and pancreas. And then to talking about the small intestine on.
That's the reason I picture Steve Jobs up is many of you will know that he died from pancreatic cancer,
but he seemed to be in the news about his pancreatic cancer for many, many years.
And if he truly had a sort of pancreatic adenocarcinoma, he would've passed away much more rapidly.
But he actually had pancreatic net, and that's part of the reason he lived for so long.
I gather he didn't even have conventional therapy for that. So as I said, the very fact that they're rare, but they're also very, very slow growing.
So these this table shows the five year survival rates for nets with both local and distant disease today.
And we're talking about our small intestinal nets and you can see that even if you've got liver metastases with just standard of care treatment,
there's a 54 percent five year survival rate. And this impacts very extensively your clinical decision making,
and you can see that these vastly outweigh the equivalent survival rates for patients who've got adenocarcinoma with distant disease.
So what do they look like? So this is a picture from a publication showing a typical net in the small intestine.
I'd say that actually, in my experience, a lot of the primaries are much, much smaller than this,
and you can't often even be able to detect them from the outside the small intestine.
The only way you can actually identify them is through by manual palpation, and they are often multifocal.
So you will have met acronis tumours throughout the ileum that you can detect by by Mannu, by, by Emmanuel palpation.
You might think, Okay, well, that's pretty easy to take out, and these primaries are not the problem surgically.
The problem is the mes and taric spread, and this is a resection specimen,
not mind of a patient to use had the primary MI, but also with the mesenteric lymph node spread.
And you can see here that all of these small bowel loops have been pulled into this and secularised into this large mesenteric deposit,
which has significant plasma plays in making the resection of it challenging.
It's also not uncommon to come across this upon opening up the abdomen and you find bowel that you
would never leave in if you were closing and you think the patient was going to wouldn't survive.
But they have chronic ischaemia with and you open up and you see that the boundless,
unbelievably unhealthy and this chronic ischaemia causes significant bowel thickening and subsequent obstructive like symptoms.
These tumours are really rare, so they, if you take in context about colorectal cancer, is going to affect what between one, 14, one and 15 of us.
These occur in about one in 100000 patients. And because of this very slow and indolent growth, they often present very late with metastatic.
Spread or mesenteric deposits? We probably all remember carcinoid syndrome from medical school, and they can also present with those symptoms.
The the treatment of them is is complex and requires a very well-functioning MD.
Clearly, surgical resection is is is the mainstay of treatment with curative intent.
But also involved is somatic statton analogues such as octreotide and unretired and for liver disease,
radiofrequency ablation, embolisation liver resection or even indeed, liver transplantation.
Chemotherapy is only really of benefit when these tumours differentiate and start to become rapidly
prolific because of the nature of chemo only really working on cells that are rapidly dividing.
So the science, though, is absolutely fascinating, so unlike colorectal cancer or other adenocarcinomas, we know so little about these tumours.
We don't know what the cell of origin is. We don't know what the driving mutation is.
We don't know what the what the aspect of epigenetics is on that development or how these tumours evolve and progress.
And there's lots of very low hanging fruit related to the science of these tumours
that make them very attractive to study a little bit more clinical detail.
Briefly, on how these tumours are present.
So they said they present late, they have obstructive symptoms rather than problems secondary to the primary primary.
The obstructive symptoms are secondary to ischaemia. I would say that live in actual pain isn't a common way that these patients present,
but I have had one or two patients who presented with liver capsule pain and deranged liver function tests,
which are then subsequently be investigated and found to have metastatic disease.
And this demonstrates the characteristic flushing associated with carcinoid syndrome.
We use enacts guidelines to manage, work up and develop subsequent follow up pathways for all our patients.
And if you're interested in that and very much strongly recommend looking at these guidelines, which are available online at the link below,
they need to have a baseline Cro-Magnon and a which is one of the best serum
tumour markers to identify for patients for to might develop further recurrence.
A 24 hour urinary five hydroxy in daily acetic acid is a prerequisite to identify and make the diagnosis.
Staging radiological is key. But most importantly,
in relation to identifying the anatomy of the vasculature that supply the the primary as well as the mesenteric
disease octreotide scan is very important to identify occult peritoneal disease or liver metastases,
but cannot on occasion be equivocal. And in those situations, a pet scan can sometimes give you the key in relation to surgical work up.
An Echo is also important to identify or call carcinoid valve disease, which is again is not uncommon in these patients.
And as I alluded to earlier, it's very important to have a functioning MDT with all of the associated subspecialty support.
Staging is really quite simple. There are different staging types that have been put about.
These are the innate stagings and as you can see that a tumour stage is based purely on size
and invasion and the Anandan binary zero one and you have your stages of zero to four.
So very straightforward staging. It doesn't really help, though, in the management, to be perfectly honest.
So the general principles,
as with any sort of solid organ malignancy for if they haven't got metastatic spread and they have limited nodal spread the game for for curative.
The aim for curative resection is radical resection with purity intent.
So resection the primary and radical launchpad.net to me to remove those mesenteric lymph nodes that are involved,
there is some discussion as to whether this is safe to be done laparoscopically or open.
The current recommendations and what I would agree with is that for anything other than respecting
somebody who's got limited primary and mesenteric disease and they have metastatic disease in the liver,
then only in that situation is a laparoscopic approach appropriate.
And for all other approaches, there's this should be done via an open resection.
And that's partly so you can make sure you get out all of the Mes and taric disease,
but also due to the multiple Scality of the primaries and various techniques have been
looked at imaging and endoscopic to identify whether to pick up these other primaries.
But it's been shown that only by manual palpation has the highest sensitivity to pick up other primary,
so you have to do it open if you've got metastatic disease.
Things become really quite complex, and it's partly related to the tumour biology and how slow growing these are,
and you have to tailor what treatment you're going to be offered to,
how long the patient's likely to live with those disease, and they can live for many, many years with relatively stable liver disease.
So there's various options for the liver, as I mentioned, and there are various palliative options for the primary the mesenteric disease.
If these patients have obstructive symptoms, chemo, as I mentioned, is possible.
Patients have highly proliferative disease,
so this patient on the right is actually a consultant colleague who I operated on about three or four years ago,
and she had a palliative laparoscopic right. And you can see she's got very extensive liver disease.
She's now had stable disease, I believe, for about three years.
And if that continues, then she would potentially be a candidate for our first liver transplantation for metastatic net.
So this is a sort of, I guess, one of the key slides in relation to the surgical decision making,
and these are the questions that go through my mind when I see a patient in clinic who I'm trying to work out for,
whether they are, they can be resected with curative intent.
So as with any patient, he first see, the key thing is, are they fit for surgery and are they going to engage with the process?
And I think patient engagement with the decision making process is absolutely key.
They've got to buy in to all of the disasters that are associated with radical mesenteric resection.
They have to understand, albeit there's only a small chance of it.
If you if you work at these patients correctly, that things can go wrong in the operating theatre, they could end up with shortcut.
They could end up with a high output judge, an ostomy. They could end up on lifelong pain.
And I don't think that you should embark on these operations where you're high up
on the asthma and asking the if the patient hasn't bought into that whole process.
So the other question is how symptomatic they are from their primary and these enteric disease.
And then the question is whether or not this is respectable.
Some studies have shown that if you there's invasion of the mesenteric disease into the retroperitoneal structures such as the duodenum,
then that's a contraindication to surgery. I wouldn't say that's necessarily the case.
I think there are patients as one to talk about next, where you can go ahead and perform limited duration activity associated with the resection.
You can't pick up the doesn't apply to very easily on imaging. So that needs to be sort of taken into account.
But the key bit here is that in relation to the number of proximal branches of the estimate that that are spared.
So this is the Uppsala staging system, which I lifted from this chap's PhD thesis and the nice drawing he's got.
And they describe these four stages of mes and disease one to four.
Again, I think this somewhat oversimplifies the process, and I think you have to have a tailored, personalised approach to each each patient.
Generally, as a rule of thumb, if you have three spare judging branches that should leave you with 100 centimetres of small bowel left.
But apparently, these are the emoticons for grimacing. I didn't say that, but it can be.
It's not always. It's not always an exact science.
So you so ideally you probably want more of those, but anything less than three,
then I think you're on a hiding to nothing with relation to not having enough bowel related debunking,
which is something we shy away from in our bowel.
Adenocarcinomas can be used for patients who have got larger disease,
so very much applying the principles of going to oncology to an intestinal tumour.
So I'll give you two examples of patients who we've operated on just to sort of give you a flavour of some of the
difficulties and in relation to clinical decision making in relation to patients in the surgical approaches that we use.
So this this first patient was a 70 year old gentleman who presented initially
with urological symptoms and then went on to have imaging for his haematuria.
And he had a primary which have highlighted this is this amazing terek deposit in red.
The primary was in his ileum, and you can see the bowel lips are tethered in some fat surrounding around the median direct deposit.
And then green is his duodenum, which is of tented up, potentially involved in the and Tehrik deposit he had.
He was pretty worked up quarantine ATS guidelines.
He had his urinary levels measured and he would start on unretired, but he had no metastatic disease.
And given that he was getting obstructive symptoms, we we looked at him with a view to radical resection.
So I mentioned before that the key aspect with these patients is identifying the arterial and venous anatomy associated with the deposit.
So this reconstruction of the SMA demonstrates, you know, you can see that the the the vessels here,
the distal part of the asthma are all being pulled in to the tumour and the tumours have just highlighted by this little area of calcification.
The tumours don't appear very clear on on on CT.
This is the same imaging from the same patient cross-functionally and on the right.
What I've drawn on here is highlighted actually the area that was involved by the Meis and Tehrik and primary deposit,
which basically were just one in the same.
And you can see here we got one to three, possibly four, but that's probably going to go in the resection margin.
So three three judge panel branches and a duodenum that was involved, but we deemed him suitable.
After getting a second opinion from Southampton for radical reception.
So what are the what's the surgical approach these patients towards the latter end of my my training?
I was fortunate enough to go to visit this gentleman and along and in Bavaria, which is a really beautiful part of Germany,
I must say so it's a lovely place to go to this lovely university city outside of Nuremberg.
And this chap here unusually tall and not that I'm small, he's unusually tall,
is is is a chap called Verna Hamburger and he's the head of the department.
And he developed this technique called complete musical excision, which is a very and he used it for adenocarcinomas of the large bowel.
And it's a much more radical limp anatomy and a very close, sharp dissection along Ember Logical Planes.
And he's published his outcomes in in various journals and improved five year survival rates for patients with,
even with early stage, the old school duke's achievements to approaching almost 99 percent.
It has come under some criticism more recently because of the morbidity associated with the procedure,
but it was extremely useful to go out there and see him operate and start to learn the technique.
So the technique that we use is applying say a bit for these NAT resections.
And these two images from one of Hamburg is descriptions of Sammy describe the essential steps.
So here you've got the caver in the aorta and this is the duodenum, not the Koran.
So you do a very radical full cocker ization of the genome to bring it up out of the the wound so you can access on the right
the root of the Atom A and the assembly to identify your whether you can actually reset this and leave enough bowel intact.
So we mobilise everything. And then we go, obviously go above to then get access to this area here.
So next case is going to describe this is again,
sort of shows some of the interesting intraoperative decision making that you have to make and the importance of working well with colleagues.
So this was a 63 year old lady who presented with typical features of carcinoid syndrome.
She was investigated by our numerology colleagues and was found to have multiple liver lesions.
Of note, her urine levels were extremely high. So that's a very high five level and again, a very high associated Cro-Magnon level.
She got obstructive symptoms, however, from her primary and her actual primary, mesenteric disease was relatively minimal.
But you can see on this opposite side scan, she's got significant liver disease.
So because of her obstructive symptoms and otherwise good health, we plan to do a palliative laparoscopic right.
Amick Let me say she because of our high, high levels.
We gave almost all our patients have this. They have an octreotide infusion Perry optimally to reduce the chances of a carcinoid crisis happening.
We gave her a bonus, followed by quite a high infusions.
This is 10 times higher than what people have previously described as the infusion required and based on current literature,
the incidence of carcinoid crisis with this degree of octreotide infusion is only about two percent.
You can see what's going to happen. She got converted due to plays laser.
As soon as we started manually handling the tumour,
she had a pin drop to arrest and we felt that she'd had a customer crisis looking under the drapes.
Everything a skin was bright red. The endocrine colleagues came into theatre and were in complete agreement.
She'd had to interrupt her carcinoid crisis. So we were then left with this very awkward surgical dilemma of an otherwise fit
lady who has had an arrest on the table would mobilise the bowel by this stage,
but hadn't actually divided any vessels or got the tumour out with obstructive symptoms and really imminently about to obstruct.
So what options were available? We could take her to ICU, bring her back, then stabilise her, bring her back the next day.
But the concern was as soon as we start mobilising this tumour again,
we're going to be in exactly the same situation we could the function of bring a loop out upstream of the primary,
which is one option we could close and not do anything and just stick around assays and treat it punitively.
So lots of things to think about and lots of discussion with with with colleagues,
with endocrine colleagues, with anaesthetist, with critical care support. And in the end, we did that.
We did a very quick smash and grab right Hemi colectomy and got the tumour out within five 10 minutes.
With with with relative stability and obviously didn't join her up and gave her an end.
I lost me and she actually did very well following this, and she's gone on to have treatments for her liver disease.
However, the moment this person, this patient crosses the threshold into Addenbrooke's, she seems to develop a carcinoid crisis,
you seen that even looks at like a medical instrument, and she has a constantly crisis.
They try to do an RFA on her, and even with the local anaesthetic, she started about constantly crisis like symptoms.
So she sees me back in the clinic and she's desperate to be joined up, but I'm less keen.
So in summary of our clinical experience, I'd say that is really key to work as a team.
You need a dedicated and experienced in these tests. You need a lot of endocrine support, octreotide infusions and infusions.
And really, I think open surgery is the key unless you're doing a palliative, a section with minimal disease.
We use CMMI resection temporary slinging of proximal vessels to adapt to be absolutely certain that you're not going to lose a lot of small bowel.
This is my friend. The ligature impact I think it's really useful for for with the decimal places in the country,
and you also need to have, aside from the ligature, you need to have good friends available for when things get hairy.
So moving away from this, well, moving away from the clinical side,
I'm now going to just touch on some of the biology, which I said is really fascinating.
And if there's anything you don't understand to ask me, it's all really unknowns, as I alluded to earlier,
and this is partly due to the paucity of tissue for analysis and how rare these these tumours are.
So most of the larger scale studies have only really been approaching around about 100 patients.
The genomic studies of the very early ones identified that there was some somatic copy number aberrations and there was a loss of chromosome 18.
But interestingly, when they looked at chromosome 18, where the usual suspects, such as samat four are located, none of these genes have been mutated.
The probably the biggest study, which was this one published in Nature Genetics,
which used whole exome sequencing on a panel of 100 small intestinal nets identical in the keys.
In the title, they suggested that CDK scan will be with a was a commonly lost trunk or mutation in nets.
But actually, when you get into the details of this paper is not common,
and it was only actually present in around about 10 to 15 percent that tumours.
So I wouldn't say that it's a it's a driver of of nets the the role of epigenetics,
but methylation histone modifications microRNAs is largely unexplored.
There have been a couple of studies that have looked at methylation identified this global hypermethylation associated with nets,
but again, no specific genes and microRNAs.
Like most microarray papers, they come up with a list of four or five that they think are involved.
But really, that's that's about it. I've been interested in it in relation to the phenotype associated with nets and that the characteristic
secretory phenotype of the hormones and the relative quiescence of the cells that are present.
And in particular, we've been interested in trying to understand what the cell of origin of nets are.
And this paper from this this American group was published in Gastro about three years ago,
and shows give some very interesting circumstantial data, which agrees with what we think about what the cell of origin of nets are.
So I'm just going to just show you one of the figures from that paper.
So this is some immunohistochemistry and in situ hybridisation, which of two imaging techniques to identify what genes cells are expressing.
And here's your team on the light, right? And they've got tryptophan hydroxylase staining in dark blue.
And here they've just countersigned it in a red will change the colour to a red.
So you can see that this is a crypt which is present in the in the tumour, and they've got high tryptophan staining,
as you'd expect in the net, but also in association with this in the same cells.
You've got very high expression of algae, all five and algae. All five is this ubiquitous stem cell.
Marcus, if you're a stem cell biologist, the moment you hear out your five, your ears prick up.
The conclusion from this paper was that they felt that these these familial nets arose from a subset of intra chromatin cells,
which are essentially enter endocrine cells that express reserve intestinal stem cell markers.
So this is this is very much in agreement with some of the data I'm going to show you next.
So I can show you a mixture of published and unpublished data.
But before we go into the details of it, I'm just going to remind you of the cellular makeup present within the gut.
So within the small intestine, we have crypts embedded into the wall of the bowel.
We are villi projecting into the lumen of the bowel colon exactly the same.
There are no variety within the bottom of these clips is the stem cells, and which we known as the stem cells aimed for donkeys years,
30, 40 years and within the bottom of these, there are some undifferentiated cells, which are these slender green base columnar cells.
There's also an undifferentiated, very slowly cycling cell termed the labour retaining cell in this characteristic +4 position from the
bottom of the crypt and then cells that are also present in the bottom of the secretary Paneth cells.
And these are only rarely found in the small intestine in the colon that you occasionally find pretty,
that they're very essentially uncommon in the colon.
And then you have absorbed of anthracite and lots of secretory other secretary cells, a goblet, cells, top cells, an enter underground cells.
And these are the cells that are really very prominent within our necks.
So, so what is a stem cell in the small intestine? And this is the seminal publication from Hans Cleves, this group who's based in Utrecht in Holland,
and he used a technique called lineage tracing to very clearly identify the age of five.
Mark was was the bona fide mark of homeostatic stem cells in the gut and lineage.
Tracing is a technique which you do in mouse models or animal models,
and you develop the animal model whereby you give the mouse an injection of a drug and that drug cause the
activation of an enzyme and that enzyme cuts and pastes in your DNA or the DNA of the cell that it's activated in.
And you can cause it that enzyme to be activated only in specific cells because enzymes driven by gene reporter.
So you, let's say you're interested in saying, is our five a stem cell marker?
You're on the back of the algae, all five gene. You get activation of this enzyme called CRE recombination only algae.
All five expressing cells activate the enzyme.
The enzyme then cuts and pastes and causes the expression of a of a reporter that you can actually visualise now because that's a cut and paste.
So it's a permanent genetic rearrangement or the daughter cells of that cell will inherit the activation of the reporter.
So if it sounds a stem cell,
then the daughter cells and the cells of the daughter cells and therefore they're on will all activate will express the reporter.
And if you then stain for it, you'll see clones of cells expressed in the report and that will show that there is a true stem cell.
So that's the technique that Cleaver's used, and he very clearly showed that indeed, these ourjob five cells were stem cells.
So the question was what what are these other cells? What are these?
The undifferentiated, slowly cycling cells in various publications suggested various markers BMI one empty at Hop X and Hourigan,
as marking these undifferentiated, slowly cycling cells, which might be more likely to be the the cell of origin of the Nets.
And Cleaver's came back and then said, Well, actually, that's all well and good, but all of your other markers actually overlap with our Channel five.
So in fact, you're just describing our original five cells.
So rather than using the technique the Cleaver's used,
we decided to approach this problem in a different way by isolating the slow cycling cells based upon the nature of them being slowly cycling.
So we developed this mouse model where you've got expression of yellow fluorescent
protein tied to a histone that's driven from an intestinal specific promoter,
but as a pulse of expression. So here's a cross-section through the mouse gut.
We've given the mouse, the injection and all of the anti-north.
All of the cells of the intestine epithelium express the yellow for protein, but it's just a pulse of expression.
It's not on all the time. So with time, as the cells migrate off the villi and are lost or apoptosis and died, the signal becomes lost.
But by three weeks, you can see at the bottom of the crypts there are still these so-called labour returning cells,
which retain the label and are therefore quiescent or dormant and haven't divided.
And then you can use various techniques to pull these cells out and interrogate them for what genes are expressing,
whether they grow in culture and the like.
And we, in collaboration with a group who are now based in Denmark, we could pull these out using two other a combination of two of the markers CD24,
which marks the bottom of the crypt and a lectin called EULEX leptin, which would rule out us pulling these long Paneth cells.
You might remember I mentioned earlier on,
so the combination of these two markers allows us to pull out our labour, retaining cells with very high affinity.
So here are some actual images of the cells that you can pull out here on a
label retaining cells which are CD24 positive wifey positive leptin negative.
Here are the rapidly cycling cells because they've lost in the bottom of the crypt
that see 24 positive wifey negative leptin negative and then the pain of the cells,
which are double positive for the Moon. And as a result of using this mouse,
we we had sort of three different publications describing the nature of the labour retaining cells and actually what was happening in the bottom,
the Crips. And to summarise what we read about, so eight years work on one slide, we were able to show that indeed these.
al-Aziz expressed LG five. But they also importantly expressed this other marker called PEG.
Three or one? They fell into a population called the site population, so the site population on this graph is marked by this, by this black box.
And you can see all the cells present in that grey colour fall into the box.
And the side population is a technique that's been used in blood to identify stem cells in the blood.
But it's also clinically important because it's the same population is shown to overlap with the extrusion of DNA binding dyes,
so thereby chemotherapy resistance that they are potentially of interest from a clinical perspective.
We found that during normal homeostasis, these cells weren't stem cells, and they were actually enter endocrine and Paneth cell progenitors.
And we did this by using short term lineage tracing. I won't go through the details in this graph, but you have to just take my word for it.
But interestingly, when we put these in culture into organoid culture, which is when you can grow sort of mini organs, in addition three dimensions.
These cells were able to then grow into organoids, implying they had some degree of stem cell potency still latent.
And then we finally resolved all of this by generating another mouse model whereby we could lineage trace,
as Cleaver's did but direct, based for the first time in the mammalian system,
based upon how long a cell lived and were able to show that indeed, when you injured the epithelium and this in the epithelium as generating these
cells changed fate from being a secretary progenitor to being a stem cell.
So that was all very good. We've gone on to collaborate with a group in Barcelona led by Adwa Patil and identified
two new markers of slowly selecting cells in the colon and the small intestine.
With that whereby seven and Max 3a, they can both generate multiple image clones.
And they also both form organoids high efficiency.
But the interesting thing about our labour retaining cells is the relationship to enter endocrine cells,
and this red markers Kramer in which is remembers the serum marker we used to,
you know that patients and these labour retaining cells are destined to become endocrine cells, as well as the Paneth cells.
And more recently, after our publication, there's been about sort of six or seven other publications describing similar features of cells.
So cells that are destined to become one type. But if you push them and injure the epithelium, they then acquire stem cell capacity.
But of all these other populations are, alas, these are the only ones that are entering into crimes.
Our progenitors and combined with the very slow cycling dormant state, leads us to believe that they may well be the cell of origin of gnats.
So are they? So this is unpublished data. Here are our losses.
First of all, I mentioned that P.W. one was a very specific marker of the labour retaining cells.
And indeed, we've gone on to validate that in lots of ways and become very interested in P.W. one, which is also known as Peg three.
It's an imprinted gene. The P.W. an expression overlays very nicely with with our alliances and looking at the whole gene transcriptome data.
So looking at what genes or the genes that are expressed by our labour retaining cells and then comparing it to previously published datasets.
This is called gene set enrichment analysis and the fact that this Green Line is all
pushed over to the left hand side and you can see the bunching up of the black hair this.
This is basically overlaying all our genes, which are the signature in our labour retaining cells amongst a previously published
data set of enter endocrine cell progenitors showing that indeed they have very,
very similar gene expression profiles.
This is some single cell data whereby I'm identifying where cells that express PD one lie in relation to all the cells that are present in the gut.
These are the enter sites which I haven't highlighted, and then you can pull out genes in these single cell data.
So these are individual single cells where these different populations lie, and you can just see up here.
All of the red dots are present within the enter endocrine cell pool,
showing that the P.W. one cells are indeed enter into growing and in the end trend declining cell population.
But in fact, they ended. They're actually within the progenitor population subpopulation of this group.
So if you then take it to nets and then you say, well,
all the genes that are expressed in our labour retaining cells overexpressed in nets, you see the same thing.
So genes, that enrichment analysis demonstrates an enrichment of the signature within nets and turning that question on its head,
looking at all of the genes that are defined that define the gene expression profile of nets.
Again, these are all overexpressed significantly within our labour retaining cells.
So we have a very strong suspicion that the labour retaining cells are indeed the cell of origin of the Nets and the finals of science lead is also.
One is of real interest because it doesn't just appear to be a marker of introductions out progenitors.
It seems to control their fate. So we've got a knockout mouse from a collaborator in Paris.
These are the control slides, so this is one expression. It is dark.
You have to believe me. It's dark right at the bottom where the alliances are. There is some low level expression on the fly,
and we can knock out the expression of it using tamoxifen to to eliminate the gene and doing transcriptomic data.
So looking at what genes change when you knock out the gene? What happens is you lose all of your mic targets,
which is basically saying that everything stops proliferating and you lose progression through the G2 m checkpoint of the cell cycle.
So again, things appear to slow down. This is validated using some staining for the RTU, which is basically a synthetic nucleotide,
which we can give to the mouse and is incorporating cells that actively dividing.
Here's the quantification of that, where you can see that in the wild type mouse,
you've got higher levels of biology and corporation compared to the knockouts.
But what is most fascinating is that when you lose expression of P.W. one that a large number of the cells change into an trendlines cells,
you get much higher numbers of Cro-Magnon positive cells throughout the crypt, Phyllis Axis and also tuft cells as well.
So it's a very interesting gene. It's unfortunately very difficult to work with because it's expressed at very, very low levels.
My collaboration Paris has had three goes at trying to generate a mouse model where we can lineage trace from the P.W. on cells, all unsuccessfully.
But we're having another go at that ourselves, but it's a particular interest.
So the summary of where we are with the net biology, we're still very early stages.
They the clearly have shown the the Aloisi work is very well published and very clear,
but the work with PD one is still somewhat early and ends in its stage.
But I think it's a very it's a very interesting gene, largely unexplored in the gut.
So what are the future directions for net work in in Cambridge in relation to the surgery and the science?
We're in the process of applying for a centre of excellence status. As I said, I've got a colleague who's joining the department next month.
[INAUDIBLE] be I'll be training up on net surgery and will be joining me in helping out with getting through all our cases.
I mentioned we're going to hopefully try and generate from the scientific side
this P.W. on creating two mice to enable lineage tracing and PD one cells.
We're generating a big biobank of organoids derived from the tumours and the normal matched normals,
and also trying to model net development by using CRISPR to target candidate genes from using this more from the small intestine.
So thanks very much, everybody for listening. Obviously got to do my my thanks.
I have to thank Doug Winton, who's my scientific mentor in Cambridge and is an absolute fantastic, world leading stem cell biologist.
My previous all raise various core facilities in my old institute colleagues,
most importantly patients for their samples and consenting to our studies.
Ruth Casey, who leads on Team Paul Roe, who's my anaesthetist for the difficult nets.
Simon Harper, who's the ah, HPV net surgeon who we often do combine cases with and my various collaborators throughout the world,
and funding bodies Cancer Research UK and asks for funding my fellowship on Christmas Eve.
Core funding support from the Wellcome and the MRC at STEM Cell Institute.
Thank you very much for listening.