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Abstract

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Get to know Kai

Kai Kretzschmar, PhD

Postdoctoral researcher, Hubrecht Institute, The Netherlands

Kai Kretzschmar, PhD

Biography

Kai Kretzschmar received his PhD from the University of Cambridge (UK) in 2014. During his doctoral training under the supervision of Fiona Watt, he studied the role of canonical Wnt signaling in the plasticity of adult murine skin and epidermal stem cell heterogeneity. Subsequently, Kai joined the laboratory of Hans Clevers at the Hubrecht Institute in Utrecht (The Netherlands) to investigate the heterogeneity of the tumor microenvironment in colorectal cancer.

Learn about Kai’s research

Title: Investigating colorectal cancer using epithelial organoid cultures

Learning objectives

  • Understand the advantages of epithelial organoids cultures for (colorectal) cancer modelling
  • Learn about applications of (colorectal) cancer organoids for experimental, diagnostic and therapeutic use

Colorectal cancer (CRC) develops during a multi-step process from small lesions of the intestinal epithelium. Genetic mutations in the canonical Wnt signaling pathway are considered to be the initial step of tumor formation. Using 3D organoid technology, tumor organoid cultures from CRC patients can be established. CRC organoids closely recapitulate key properties of the original tumor epithelium, including histological appearance, general gene and protein expression pattern and mutational load. Tumor organoids are amenable to radiation treatment, gene-drug association studies and high-throughput drug screens. Organoid cultures from adjacent healthy colon mucosa that retain the identity of the healthy intestinal epithelium in vitro can also be generated. Sequential introduction of the most commonly mutated CRC genes (APC, P53, KRAS and SMAD4) into healthy colon organoids using CRISPR/Cas9 genome editing technology allows for the modelling of tumorigenesis in vitro. Xenotransplantation of mutated organoids into mice recapitulates critical features of CRC progression and metastasis. In sum, (cancer) organoid technology can be used as experimental tool for basic research as well as diagnostic and therapeutic tool for (personalized) medicine.

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Presenter:  Kai Kretzschmar, PhD Postdoctoral researcher, Hubrecht Institute

0:00:01 – Slide 1

Hello everyone and welcome to today's live broadcast, Investigating Colorectal Cancer Using Epithelial Organoid Cultures presented by Dr. Kai Kretzschmar, Post Doctoral Researcher Hubrecht Institute.  I'm Alexis Corrales of Labroots and I'll be your moderator for today's event.  Today's educational web seminar is brought to you by Labroots and sponsored by Thermo Fisher Scientific. For more information on our sponsor, please visit thermofisher.com.  Now let's get started. (0:00:45)

Before we begin I would like to remind everyone that this event is interactive.  We encourage you to participate by submitting as many questions as you want at any time you want during the presentation.  (0:00:56) To do so, simply type them into the ask a question and click on the send button.  We'll answer as many questions as we have time for at the end of the presentation.  Also please notice that you will be viewing the presentation in the slide window.  To enlarge the window click on the arrows at the top right-hand corner of the slide window.  If you have trouble seeing or hearing the presentation click on the support tab found at the top right of the presentation window or report your problem by clicking on the answer in a question box located on the far left of your screen.  (0:01:28)  This presentation is educational and  thus offers continuing education credits.  Please click on the continuing education credits tab located at the top right of the presentation window and follow the process to obtain your credits.

I'd like to now introduce our presenter, Dr. Kai Kretzschmar.  Kai Kretzschmar received his PhD from the University of Cambridge in 2014.  During his doctoral training under the supervision of Fiona Watt he studied the role of canonical Wnt signaling and the plasticity of adult murine skin and epidermal stem cell heterogeneity.  (0:02:06)   Subsequently Kai joined the laboratory of Hans Cleavers at the Hubrecht Institute in Utrecht to investigate the heterogeneity of the tumor microenvironment in colorectal cancer.  Dr. Kretzschmar, you may now begin your presentation.

Thank you very much for this kind introduction and the title of my presentation you have already heard.  I will talk about using epithelial organoid cultures to investigate colorectal cancer.

0:02:35 – Slide 2

I have an overview provided.  I will first actually introduce you to colorectal cancer, give you some facts about it and some kind of basic biology to this disease and then I will introduce actually what epithelial organoid cultures are and how we can use them to grow normal colon epithelium  as well as colorectal cancer tissue.  I will continue in the third part of my talk showing you some data that we have published, how we can actually use organoid cultures to biobank actual cancer tissue from patients and use them for drug screens analysis, etc.  (0:03:15)  In the last part of my talk I will actually show you how we can use normal colon organoids and genetically manipulate these to study and model the progression of tumors as you would see them in a patient. Okay, let me start with an introduction to colorectal cancer.

0:03:31 – Slide 3

Colorectal cancer is actually one of the most common cancers in the world.  It's in the top five of new cancer cases per year but also actually in the top five of cancer related deaths per year and therefore, of course, a very important cancer to be studied.

0:03:53 – Slide 4

Classically it's been believed that colorectal cancer rises in a canonical way from normal colon is a theme which acquires mutations that lead to the development of a small adenoma which then progresses into large adenoma and then eventually in (inaudible) patients it presents itself as a proper colorectal cancer carcinoma.  (0:04:19)  And study by Vogelstein showed actually that these steps towards the disease associated with mutations in very critical genes such as APC, KRAS, P53 and SMAD4.  The mutations that you find in APC and KRAS actually activate the pathway so they are chronically up regulated and active.  Whereas P53 and SMAD4 are usual mutations that in activate these oncogenes and therefore allow progression towards a cancer.

0:04:55 – Slide 5

Now this classical Vogelgram colorectal cancer is actually only one of four subtypes that are nowadays studied.  There is another characteristic one which is called micro satellite in stable tumors which is the one you can see on the left. Then the next type of colorectal cancer is the so-called canonical one that I just presented.  And then there are two other types, a metabolic one and a mesenchymal one and all these four types can actually be studied in our (inaudible)  culture system.  

0:05:27 – Slide 6

Now why is it important to have a good in vitro method and what in vitro methods are there to study cancer?  Well, of course you cannot study cancer very well in a patient so you need to use some methods outside the human body.  And traditionally people use cell lines that are arrived from tumor biopsies but these have several issues.  For example, of course they don't show the heterogeneity within a tumor so they tend to grow very clonally and at some point will be representing essentially one cell found in a tumor.  (0:06:01)

There are also genetically very unstable, acquire a lot of mutations due to the culturing.  The other system that people have used is transplanting tumor cell lines or tumor cells from the patient into mice and that of course has issues because humans and mice are not alike.  The units use immunodeficient mice and therefore cannot study how the immune system for example influences tumor growth and similarly through the colorectal cancer cell lines you do not have the heterogeneity of the tumor reflected in the system.  And last, not least this system is very low in throughput and very expensive.

0:06:44 – Slide 7

So the development of organoids and I'm showing you in the bottom essentially our system, how we actually generate these.  We take a biopsy of the adult small intestine of a patient for example, or of a mouse and also the colon of course.  We can isolate the crypts which contain the stem cells and we can from there isolate the stem cells and put them in an extra cell, a matrix switch environment and grow them out into so called intestinal epithelial organoids.  There are other ways to generate organoids that I would just show.  One of them is actually directly taking the crypts and putting them into the matrigel of the BME and you can generate the same organoids.  (0:07:24)  And there are two other ways you can generate organoids but these are not effacy organoids because they also contain the mesenchymal cells surrounding.  So the rest of my talk will be concentrating only on the organoids cultures I showed you in the beginning which we call it epithelial organoids.   Now if you take a normal intestinal stem cell...

0:07:45 – Slide 8

...Lgr5+ you can see now on the screen how this actually develops over time in cultures.  Where you see from a single cell over about two weeks, the whole cell will start dividing, will grow—will generate more and more cells and will essentially generate an epithelial organoid as we have in the culture.  So this is actually what happens there.  It's a very defined culture medium as well that we have associated with it.

0:08:12 – Slide 9

So what are the characteristics of these organoids? They are three-dimensional and as I said they can be established from a single adult stem cell and they require certain niche factors that are important such as EGF, Noggin and R-spondin, but they are epithelium.  There's no amazing candle, no cells, no fibroblasts in there.  In the beginning of course are generated from a stem cell but as time progresses, the culture progresses, these organoids will also contain the different shaded subtypes found in the intestine, for example enterocytes.  (0:08:49)  And they can be expanded indefinitely and in contrast to the other methods I introduced to you earlier, they are genetically very stable and actually only acquire mutations at the cellular rate as you would find in the human body.  Now we source to use this epithelium organoid culture system and use this to actually maintain colorectal cancer cells as well.

0:09:12 – Slide 10

And this is what you can see here.  So we essentially take the tumor resected tissue, so usually part of the colon is taken out and take a biopsy from the tumor and take a biopsy from about 10-15 cm distance normal epithelium , make single cell preps or isolates the crypts from the normal colon and then can put them into the medium as you can see on the screen.  (0:09:37)  We can generate these organoids as I showed you earlier.  So this is our starting point for the continuing studies.  Now you could ask how can we make sure that in the tumor for example, we do not have any normal tissue left.

0:09:53 – Slide 11

Well, that's very easy because actually a last fraction of the tumors have mutations in the Wnt pathway almost 100%, so they have actually chronically activated Wnt and therefore do not require a condition medium that we use to maintain the normal colon organoids, that is Wnt condition medium.  (0:10:17)  So essentially by growing the correct cancer organoids in the absence of this Wnt condition medium we can ensure that we only grow tumor tissue.  And in the normal tissue we usually can actually identify it by a sequencing, whether it's normal or not and usually the growth will tell you as well whether it's normal tissue.

0:10:41 – Slide 12

Now we can use these colorectal cancer organoids in the adjacent normal tissue to generate a biobanks by essentially taking a lot of biopsies from patients that gave informed consent and we have created a foundation that actually takes care of biobanking this, and then you can see on the screen several other tissues and associated cancers for which we have established living cancer organoids biobanks.  And what do we do with this? (0:11:06)  We can use these organoids and these biobanks as an experimental tool where we can ask basic biological questions related to stem cell biology, infectious diseases or cancer.  And indeed we can also use these to test drug efficiency, do genomic analysis, predict what drug might be used. (0:11:26)   So for diagnostic purposes organoids can be used as well.  And lastly, this is something that will still take some time until it's possible although proof of principle experiments have been done already, the biobanks, these organoid biobanks can be used for therapeutic use for example in the sense of transplanting organoids grown up to organ size or near organ size or at least to contribute to parts of the organ and we can use them also for gene correction, transplanting gene corrected tissue back into the patient.  But this is still something that we need to improve.

0:12:03 – Slide 13

Now I will introduce you to our colorectal cancer organoid biobank that we have created three years ago which actually is a biobank of 19 patients and their organoids grown from their tumors and we have used this in various ways.  We analyzed the DNA sequence so we could identify the tumor mutations present. (0:12:26)  We studied the transcriptome of these organoids and then performed drug screens and other things to see whether we can actually use the gene expression data to predict if particular drugs work on the tumor.  And this we can also do a comparison to the normal organoids, the colon organoids because we have those also in the culture.  Now if you don't believe me I will show you some slides...

0:12:51 – Slide 14

...showing actually that the organoids that we grow from the tumor epithelium very much reflects the epithelium in the original biopsy so you can see some endotoxin and eosin staining that show the way the tumor grows as organoid it's very much like what you see in the patients or the histological analysis suggested.  So to show this to a pathologist they really, really recognize the resemblance of the organoids and resemblance towards the patient's original tumor.

0:13:25 – Slide 15

What we've done in addition is we looked into how similar this genetic operations and mutations are that we find in the tumor and then in comparison to the organoids.  And we could see that the overlap between the two, between the original biopsy and the organoid is very, very striking.  It's close to 90% in many cases, so showing that the organoids really, truly reflect what you find in the tumor was seen in the patient.  (0:13:54)  And of course we also find the typical mutations that are introduced to you in the beginning.   APC on the top right, TP53, KRAS are typical mutations founds in colorectal cancer and they are also present in our organoid biobank and also in organoids collected or generated from patients collected late, patients almost collected a bit later.

0:14:17 – Slide 16

Now these tumor types I mentioned in the beginning can be identified by doing transcription analysis. That's what we did also with the organoids and I'm not so sure if you can see this on this screen, but if you look in the top bar you can see these orange dots and they actually represent the organoids that we have generated and their gene expression.  And you can see they fall into the four types of colon cancer, so they really, really represent the whole spectrum of colorectal cancer.  Now in addition based on for example the gene expression...

0:14:59 – Slide 17

...pattern and the DNA sequencing we can make predictions where a particular drug that targets a particular signaling pathway will work on the patient. And you can see on the top Nutlin-3a is a drug that actually will act on cells that have still normal P53 and you can see that there the IC50 of those cells that still have a normal wild-type P53, it's much, much lower than in adults that actually have a mutation in P53 suggesting that a drug that targets this pathway, will of course only work best in patients that still have a wild-type P53. (0:15:40)  And those that have a mutation in this pathway will probably not have a therapeutic benefit.  The same is true for Cetuximab which acts on the EGF and KRAS pathway.  You can see that those that have a wild-type KRAS still show a good response whereas those that have a mutative KRAS are not sensitive to this drug anymore. So if we can just generate these organoids and then actually look in their gene expression pattern we might be already able to predict where the particular drug used on the patient will actually work or not.  Now culture conditions are very critical and this is what you can see...

0:16:23 – Slide 18

...on the next slide.  That the normal epithelium is very much dependent on all these key niche factors such as Wnt and Rspondin, EGF, P38, Noggin and TGFBi, whereas the tumors are more that's actually able to grow independent of Wnt and Rspondin for example, but also other components because they simply have been patient in the KRAS pathway, therefore they will not require EGF and other signaling pathways and therefore will not require any of the other drugs. ( 0:16:56)

Now I will summarize this by playing a video that will follow.

"Organoids are longtime coaches derived from diseased or healthy tissue of patients or animals.  This can be done for almost all patients and for virtually all organs and with regard to different diseases such as hereditary disorders and cancer.  Both health and diseased tissue is isolated from an organ.  These small tissue fragments are placed in culture.  The isolated stem cells then grow into organoids.  These organoids are very similar to real organs and form a reliable testing environment for medicines against all kind of diseases.  Using DNA sequencing errors in the genetic code can be detected.  We can also screen multiple combinations of medicine selectively and reliably on a large scale.  Organoid technology is a powerful method which can be used to design and test patient specific medicines. " (0:18:23)

Okay, the last part of my presentation I will introduce you to ways that we have developed to actually start from normal colon epithelium and generate organoids from this that introduce mutations that are relevant for cancer progression and then see how things change in the organoids and I introduced you in the beginning already these four key mutations that are found in the classical colorectal cancer, APC mutations, KRAS, P53 and SMAD4.  We can actually replicate this by starting from normal tissue and then see what happens.

0:19:02 – Slides 19

We use CRISPR/Cas9 for this which is a genome editing technique that allows us to directly introduce mutations in particular genes.  So we can generate to sgRNAs that actually target these and then when we used this, we modulated the composition of the medium that I showed you earlier and thereby select for only for example organoids that can grow in the absence of EGF in the medium.  Those organoids that have a KRAS mutation for example.  I will give you an example on this using the canonical...

0:19:39 – Slide 20

...Wnt signaling pathway.  That actually requires Wnt ligands to be active and that's something you can see on the right inside.  In the absence of the Wnt ligands there is no gene expression of the targeting found and therefore the targeting cannot be expressed, proliferation is inhibited in those cells that do not have Wnt ligands found to their receptors.  (0:20:03) In APC mutant organoids though, this pathway is chronically active.  Essentially the Wnt ligand is not required for gene expression.  So what you see on the left is actually present so the Wnt ligand is not there but actually in terms of gene expression the right inside is the case, so the beta (inaudible)  and modulator can actually bind to the target genes and express those and therefore stimulate proliferation.

0:20:36 – Slide 21

So essentially what we do is we take a wild-type organoid growth and add guide RNAs targeting the APC gene and then we can wait a couple of days.  And then we remove the Wnt condition medium and the Rspondin condition medium which is also required to stimulate the Wnt pathway.  And then after about two weeks we can pick grows that grew in the absence of these two components and then sequence them and verify this.

0:21:06 – Slide 22

And this is actually what I show you in the next slide.  So we can see that actually these organoids have friendship mutations.  So you can see, this is how it looks.  In the top left of the organoid panel you see a normal organoid growing in the complete medium and all the other five boxes show you organoids that grow in the absence of Wnt and Rspondin and you can see only those that are actually mutant, so the bottom four pictures show you organoids that grow and then the one that has guide RNA4 grows best.

0:21:42 – Slide 23

We can confirm that these, and this is a representation of the mutation found in using guide RNA 4.  There are friendship mutations which essentially inactivate APC and allow the pathway to be chronically active and we assess this using QPCR for Axin2 which is a target gene of this pathway and you can see that even in the absence of Wnt Rspondin condition media this targeting is still expressed very nicely, actually at higher levels than with it, really nicely showing that we have introduced these mutations.  And now we can use this for all other mutations found.

0:22:24 – Slide 24

And this is what I show you here, the (inaudible) slides, so APC knock out organoids are generated, KRAS activating organoids.  They don't require EGF.  Then in addition organoids that actually had mutations in APC and P53, they were selected by the addition of Nutlin and all the other combinations.  Essentially we generated organoids that contain up to four oncogenic mutations and then we could study them.

0:22:55 – Slide 25

What you can see is actually when we transplant these organoids we can see that the (inaudible)  mutant organoids that have mutations in APC, in P53, in KRAS  and SMAD4 are the ones that actually generate a carcinoma when injected into mice, so therefore very nicely replicating what you would see in patients.

0:23:16 – Slide 26

You can also see that those organoids that have four full mutations are chromosomally instable and actually also really, really perturbed and generate aneuploidy.

0:23:31 – Slide 27

What we did in addition, we transplanted these actually into the intestinal region and then studied whether they can generate metastases and also growth.

0:23:45 – Slide 28

And we could assess that those actually that have these four mutations present and are very much like a colorectal cancer, grows very, very fast, significantly faster than all other mutations.

0:23:58 – Slide 29

And also in the next slide you can see the ones that have these four mutations are the ones that are actually able to metastasize so we can actually really nicely replicate what you would see in a patient and by genetically editing normal organoids.  So starting from normal 15 (inaudible) we can really replicate what happens in a tumor in a patient in a step by step.  We can see that only those that have all four critical mutations are actually able to metastasize.  And there's a very low number of those that have SMAD4 wild-type, so the step before the carcinoma that are also able to metastasize. (0:24:38) Possibly and actually in this case this metastases actually was a contained later on as you found out a mutation in SMAD4.  So essentially it was a (clutch of co(inaudible)) mutant organoids.

0:24:50 – Slide 30

Something else we can also do of course, is we can also generate gene knock-outs in the DNA repair complex to replicate organoids that are like microcephalies in stable tumors and we can see that those mutants that have a mutation in the MLH1 which is a very critical DNA repair gene starts accumulate lot of mutations.

0:25:16 – Slide 31

And we can actually see that based on the organoids we generate we see very, very similar mutations also in patient samples so essentially we can replicate the mutations that you would find in the patient that has these microcephalies in stable cells and we can find the same mutations by just artificially introducing these mutations and we can therefore really study the tumor.

0:25:43 – Slide 32

So in summary what we can generate is we can generate epithelial organoids from normal human colon and colorectal cancer tissue.  From this we can generate biobanks that we can use for drug screens and other things and we can also genome edit these organoid cultures to study tumor progression as well as study the origin of mutation of signatures found in the colorectal cancer and other genomes.

0:26:14 – Slide 33

I would like to thank the head of the lab Hans Clevers and also several other people that are still in the lab or have left the lab that have significantly contributed to the work, and I would like to thank all our collaborators in the Hubrecht Institute inhouse and the University Medical Center, the Netherlands Cancer Institute as well as the Wellcome Trust Sanger Institute.  And I would like to also thank all the funding agencies that make this work possible.  

0:26:43 – Slide 34

Last, not least, thank you for your attention and I'm looking forward to your questions.

Moderator:  Thank you, Dr. Kretzschmar for your informative presentation.  We will now start the live Q&A portion of the webinar. If you have a question you'd like to ask, please do so now.  Just type them into the ask a question box and click on the send button.  We'll answer as many questions as we have time for at the end of the presentation.  Questions we did not have time for today will be answered by Dr. Kretzschmar via e-mail following the presentation.  So let's get started.  Our first question is:

(0:27:32)  Question:  What is the success rate of establishing these organoids from patients:

Yes, the success rate is actually in the case of colorectal cancer pretty high.  It's 90-95% of the samples we receive actually we can generate organoids from.  So it's in the case of this particular tumor type very,very good actually.

Moderator:  Our next question is:

(0:27:58)  Question:  Can single cell mRNA sequencing be performed from the colorectal cancer organoids?

Yes, that works very well.  So we started of course with organoids from the mouse small intestine and it gives very, very good results and the same is actually also true for human normal colon organoids you can get very, very good single cell transcript on data and actually for cultured colorectal cancer organoids that is the same.

(0:28:31)  Question:  For how long can the colorectal cancer organoids be maintained in culture?

This is a very good point actually.  So we have some of these in culture for two years already and they seem to be still going fine and still be very, very primary like in the way they behave. (0:28:51)  So yes, so in principle it can be a very, very long time, much longer than you can usually do with, for example colorectal cancer cell lines that tend to kind of behave more like an immortalized cell line.

(0:29:07)  Question: Are you primarily focused on the epithelial organoids or do you culture with mesenchyme as well?

So in our system we really specialize in a (inaudible) organoid so we only have the Martingel that is supporting the growth so we don't have an actual cellular mesenchymal niche but in principle that might be something that is interesting to look at, but for our purposes in particular for the drug screens we really like to see how the tumor if the epithemal itself behaves and works and don't really do mesenchyme.  So for our purposes we do not do it, but I believe in principle it is probably possible.

(0:29:50)  Question:  What is the size of the organoids needed for the transplantation in animal models?

So I think there it really is dependent on what organoids you use and what colorectal cancer you actually implant because I would say that in some cases you might have issues with the maintenance in the transplant, so maybe they, I don't know, they differentiate away or something like that.  They're not so stable necessarily.  (0:30:22)  But a particular size I'm actually—I actually don't know personally because I'm not doing these kind of transplantations, but I would say probably organoids kept for about a week should be good.  It's just a matter or more of the number of organoids that you transplant.

(0:30:43)  Question:  How do you measure the viability percentage after drug treatment whereas organoids are variable in size?

Yes, this is indeed a very good point and that's something that we cannot—we in the beginning had really trouble with and that is that we now actually filter the organoids so that they are maximum about 40 micrometer in size.  (0:31:10)  And then really look at that to make sure that we do that. What we actually do to check for viability specifically is we use an ATP based assay which essentially gives you an indication of the overall growth and then you need to of course have controls that have about the same growth efficiency, possible use the same organoid line as control to actually adjust for the differences in size and growth.  But we usually tend to use organoids that are rather small, because they can grow much larger than 40 micrometer actually.

(0:31:47)  Question: Based on the morphology and growth rate when comparing normal organoid and CRC samples, we did not detect a very obvious difference.  Do you think the in vitro culture system cannot translate the in vivo settings in some cases due to growth factors we add?

So the normal organoids that you saw in the presentation were actually cultured in medium that is more expansion medium where we essentially maintain the LGF5 stem cells that we built the small intestine or the colonic epithelium.  So if you actually change some components in that medium you would actually get them to differentiate and they would look much, much different to the colorectal cancer organoids.  (0:32:37)  I have to say though that the colorectal cancer organoids themselves tend to look really, really different depending on what type of organoids you have.  And the ones that you saw in the presentation were actually those classical Vogelgram colorectal cancers which have mutations in, for example the Wnt pathway. And they tend to be very cystic.  (0:32:57) So you can also have other types, the mesenchymal one in particular or actually also the micro satellite in stable one where the organized of the tumor is actually very dense and you don't have this hollow cyst like structure. Therefore you can really make a difference between the normal and the tumor. Let me just have a look.  I think the critical point is that also in vivo actually there is kind of a natural gradient of these growth factors, whereas in tissue culture where we grow the organoids, that's something we cannot really replicate yet.  (0:33:28)  So that would be something by engineers would enable that that somehow you could actually have a gradient of these growth factors implied in your culture system.  You might actually have really clear differences between the tumor and the normal because the tumor organoids are pretty much actually independent of these growth factors and probably could also grow in the absence of them.

Moderator:  Our next question is:

(0:33:59)  Question:  How difficult will it be to derive organoids from other tumors without clear indication of the existence of cancer stem cells?

So I hope that the question means are the tumor types that are not from the colon.  So in most cases the problem is really to find the best media composition for the organoids to grow.  (0:34:26) So you will have to start probably from normal epithelium maybe and try to get that to grow and usually the tumor will grow in that condition too, but of course you will have to be sure that you somehow can select for the tumor either by studying what mutations are in this tumor.  I mentioned for example that you can use Nutlin in case there is a P53 mutant and this way you can select for cells that still have a wild-type P53 and would die in the presence of Nutlin.  (0:34:56)  And then only the tumor that is P53 mutant would grow out. So it depends, generally it should be possible if you really work out the right culture conditions.  Cancer stem cells is another thing, because I think there is still a big debate whether cancer stem cells are such—actually exist.  For the colon I can tell you that in most cases when we passage these organoids we actually trypsinize them into single cells and actually it's maybe not 100% outgrowth but we get a very, very good outgrowth efficiency at 90%, 95% which doesn't really make sense if you think that there are really dedicated stem cells in this tumor.  (0:35:39)  If all of the cells that you have in the tumor seem to be growing out of virtually all cells I should probably caution. So that's a different question I think.

(0:35:50)  Question;  Is there a difference for the percentage of success of organoids culture from cancer patient samples and health patient samples?

So we don't get actually organoid samples from healthy patients, so of course because what we actually, in our case at least, we get the colon epithelium  from a section.  So actually certain bits of the colon are really resected out and from there we can take the normal epithelium . The only way you could have access to normal epithelial organized from really a healthy patient is probably after a colonoscopy where they take a biopsy, but usually that's not done if there is no evidence for any let's say indication of a disease because of course you don't want to injure a normal healthy clearly clear epithelium .  So all the samples we have from human are actually from diseased from cancer patients.

(0:36:54)  Question:  Have you already established a biobank and are any drug industries interested in cancer drug testing with organoids?  Is it more efficient than cell culture?

Yes, we have established a biobank.  Actually I mentioned this hub biobank in the presentation.  They actually have established I think more than 100 colorectal cancer organoid lines now. So that's already been done and indeed we have collaborations with the drug industry matching up with several companies, big companies, and also smaller companies and they use kind of these biobanks.  (0:37:32)  So actually also companies can approach to hub biobank and ask to buy organoid lines for their research and it's going very well.  It is indeed more efficient than let's say regular cell culture.  Because the colorectal cancer organoid lines really are reflecting heterogeneity of the tumor.  (0:37:54)  They don't tend to be immortalized so they stay rather primary whereas the cell cultures, the cell lines that you use, the classical colorectal cancer cell lines, they tend to be not behaving as they were when they were primary.  So I would say it is pretty good.  But what I have to caution is that like organoid cultures in general is a very, very laborious and they money intense work.  So it would be, if you compare that it really depends on your questions that you're addressing.  It might still be better for you to use cell lines because it's very, very, you need to put a lot of effort into this.

(0:38:37)  Question:  If you passage CRC organoids via single cell disassociation, are you not worried to lose tumor heterogeneity and to select for high growth cells?

So this is a very good question.  That is surely possible, although we have actually done single cell sequencing from classical colorectal cancer organoids and they tend to be not so heterogenous as you would think.  (0:39:07)  I think the heterogeneity between patients is much bigger.  There are other subtypes, for example I guess the micro satellite in stable ones which seem to be much more diverse in their mutations status and in the mutations that they acquire.  And that could be a problem but with the classical Vogelgram colorectal cancer organoids I would not be so worried.  (0:39:30)  But of course you can actually generate clonal cultures and then study as many clones as you like direct from the tumor and then see how different they actually are.  but that's of course a problem that is possible, but that problem is even worse when you study just regular cell culture to these cell lines.

(0:39:53)  Question:  What are the special characteristics of the Matrigel you use in your organoids?  Is it commercially available?

So Matrigel is generated by a company actually, there are several companies that produce something like Matrigel, also is something called Basement Membrane Extract, BME is actually very similar. That's what we mostly use actually nowadays.  The special thing about this is that this is actually isolated from a sarcoma grown on mice.  (0:40:26)  And it contains extracellular matrix from this sarcoma, from this tumor, an astroma, and actual cellular matrix proteins that really support the organoid growth.  And that's really critical.  So that the cells are in a 3D environment, not in a 2D environment and it seems to be supporting the growth very, very well.  So that's the special thing about it. Essentially it's extracellular matrix.

(0:40:52)  Question:  Do you have experience with transplanting these organoids in Zebra fish?

Actual answer, no.  And for us really it's—we have not worked with Zebra fish cells at all in that sense that we try to generate organoids from them and also of course have not transplanted them into the Zebra fish.  So we only transplant into my steps, the only in vivo experiments in this system we actually use.

(0:41:22)  Question:  Do you have biobanks of prostate organoids or only colorectal?

I believe the biobank does have also generated those from prostate organoids.  Let me just go back to the slides so we can check together.  Yes, it's listed there.  So it is actually one of the tumor types where we do have biobanked organoids.

(0:41:51)   Question:  At what ratio can the colorectal cancer organoids be split?

They grow really, really well and in most cases you can split them one in six up to one and twelve.  So you can generate a lot of cells in a rather short time.

Moderator:  All right.  Our next question is:

(0:42:14)  Question:  How often do the colorectal cancer organoids have to be passaged or split?

Well, the passaging is really dependent on your question.  If you want to propagate the organoids and keep them really nicely in cultured then this has to be done about once a week.

Moderator:  Thank you, Dr. Kretzschmar.  Do you have any final comments for our audience?

Yes, I would like to thank all of you for listening in and if you have any questions you can always get in touch with us at the Hubrecht Institute and also get in touch, for example, with the biobank, the hub biobank and see if there are any ways you can have access to these organoid lines.  (0:42:59)  But overall thank you very much for this opportunity and for your attention. Thank you.

Moderator:  I would like to once again thank Dr. Kai Kretzschmar for his presentation.  I would also like to thank Labroots and Thermo Fisher Scientific for making today's educational webcast possible.  Before we go, I would like to remind everyone that today's webcast will be available for on-demand viewing through August of 2018.  You will receive an e-mail from Labroots alerting you when this webcast is available for replay. We encourage you to share that e-mail with your colleagues who may have missed today's live event. That's all for now. Thank you for joining us and we hope to see you again soon.  Goodbye.

End Presentation: 43:48

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