Difference between revisions of "Team:Czech Republic/Practices/Interview"

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picture of interview
 
 
=Motivation=
 
=Motivation=
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In order to get professional opinion of our project and also learn more about cancer and CTC cells we made an interview with two cancer specialist. We introduced them our ideas and discussed some interesting questions. This interview was very helpful and enriching and to our delight led to agreement about our future collaboration.
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[[File:Czech_Republic_interview.jpg|400px|right|border|Interview]]
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To get a professional opinion on our project and to learn more about cancer and CTCs, we conducted an interview with two cancer specialists. After the introduction of our project idea, we discussed some particularly interesting points. We received recommendations that helped us to improve our project. This interview was not only helpful and enriching but also led to an agreement to collaborate with the Laboratory of Tumor Biology in the near future.
  
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[[File:Czech_Republic_interview.jpg|70px|Scheme of the STE2 ORF]]
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=Key outputs=
  
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=People=
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A suggestion to use a color change instead of the agglutination as an indicator of CTCs' presence. We modified our experiments in module 2 and used GFP as indicator of successful communication between IODs. We are definitely going to extend the use of color indication in further research.
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==Mgr. Pavel Pitule==
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'''
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 +
A recommendations to test the agglutination on a slide. We have the possibility to perform our experiments in microfluidics. We appreciated this advice and used microfluidics in many of our agglutination experiments.
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= The experts =
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<html><div class="info-box"><div class="left"></html>
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==Mgr. Pavel Pitule Ph.D.==
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[[File:Czech_Republic_interview_Pitule.jpg|150px|left|border|link=|Interview]]
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Mgr. Pavel Pitule, Ph.D. is a junior researcher at the Biomedical Center at the Faculty of Medicine in Pilsen. He is the research group leader of the Laboratory of Tumor Biology.
 +
<html></div><div class="right"></html>
  
 
==Mgr. Pavel Ostašov Ph.D.==
 
==Mgr. Pavel Ostašov Ph.D.==
 +
[[File:Czech_Republic_interview_Ostasov.png|150px|left|border|link=|Interview]]
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Mgr. Pavel Ostašov, Ph.D. is a researcher at the Biomedical Center at the Faculty of Medicine in Pilsen. He is a member of the Laboratory of Tumor Biology.
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<html></div><div class="break"></div></div></html>
  
 
=Interview=
 
=Interview=
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1: My name is Pavel Pitule, I work in the biomedical centre of the Faculty of Medicine in Pilsen and our group is focused on cancer biology. We are looking for some biomarkers for the prognosis and prediction of colorectal cancer. There are two branches of our research, first one uses molecular methods such as quantitative PCR and microarray sequencing to find mutations or changes in expression in tumor tissues and second is focused on circulating tumor cells (CTCs) where we’re trying to find specific cells released from the tumor into blood stream and characterize them in regards to disease progression.
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P: My name is Pavel Pitule, and I work in the biomedical center of the Faculty of Medicine in Pilsen. Our group is focused on cancer biology. We are looking for biomarkers to use in prognosis and prediction of colorectal cancer. There are two branches of our research, the first one uses molecular methods such as quantitative PCR and microarray sequencing to find mutations or changes in expression in tumor tissues and the second is focused on circulating tumor cells (CTCs) and we are trying to find specific cells released from the tumor into blood stream and characterize them with regards to disease progression.
 +
 
  
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2: I’m Pavel Ostašov. I am working in the same group. I’m mostly in charge of the microscopy and processing of the sequencing data.  
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O: I’m Pavel Ostašov, and I work in the same group. I am mostly in charge of microscopy and processing of sequencing data.  
  
  
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1: I have just heard a little bit about synthetic biology but I haven’t used it. From what I’ve heard it can be used for analyzing blood particles.
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P: I have only heard a little about synthetic biology and have not used it. From what I have heard it can be used for analyzing blood particles.
  
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2:I know there are some drugs for therapy that modify the cell and release some toxic protein and let it find the cancer cell. And release of the toxic protein can kill the cancer cell.  
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O: I know there are certain modified cells that release a toxic protein that is able to find the cancer cell. The release of the toxic protein can kill the cancer cell.  
  
  
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2: It would be nice for detection of maybe some changes in cytokines and some differences in screening molecules and such.
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O: It would be nice to use it for detection of changes in cytokines and differences in levels of screening molecules.
 
   
 
   
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1: Maybe it could be used for detection of some metabolic products of the drug. Some cancer cells can modify prodrugs which are used in chemotherapy and then expel it outside of the cell. So maybe it would be great for monitoring the cell’s resistance to the therapy.
 
  
 +
P: It could be used for detection of metabolic products of a drug. Some cancer cells can modify prodrugs, which are used in chemotherapy and then expel it outside of the cell. So monitoring cells' resistance to the therapy would be great.
  
  
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I think you will like our project, based on what you’ve said.
 
  
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And because it touches the field of your expertise, we would like to hear your opinion and view on our project.
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'''Based on what you’ve said, I think you will like our project.'''
  
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As you know, we are working with yeasts. Normally, when you want to make some final application, you prepare your plasmids, and then you transfer them into a cell for a final product. But this is quite difficult and thus we are trying to make the assembly easier. We are using the fact that the yeast exist as haploids - alpha and a, and they can mate to produce diploids. We recognized that we can give specific properties to two haploids and afterwards mate them. The final diploid has both characteristics of the individual haploids. So it’s possible to make a library of a and alpha cells.  
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'''And since it touches the field of your expertise, we would like to hear your opinion and view on it.'''
  
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We also want these final diploids to communicate with each other. One of the diploids will produce a specific output, in our case a pheromone, and the other diploid will be able to detect it by a receptor on its surface, react and produce an output - another pheromone. The last diploid in the chain will detect the pheromone and it will in our case cause agglutination of the cells.  
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<html>
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<p class="visibility-toggle" onclick="toggle_visibility('practices-interview-long-block');">Click here to read how we described our project...</a>
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<div id="practices-interview-long-block" style="display:none;">
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</html>
  
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So how we want to do it? We want diploids to communicate through pheromones and receptors, that’s why they have to have the yeast pheromone mating pathway activated. I don’t know whether you have heard about it.
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'''As you already know, we are working with yeasts. Usually, when you want to make a final application, you prepare your plasmids, and then transfer them into a cell for a final product. However, this is quite difficult and thus we are trying to make the assembly easier. We are taking advantage of the fact that yeasts exist as haploids - alpha and a, and they can mate to produce diploids. We recognized that we can give specific properties to two haploids and then mate them. The final diploid has both characteristics of the individual haploids. So it’s possible to make a library of a and alpha cells.'''
  
 +
'''We also want these final diploids to communicate with each other. One of the diploids will produce a specific output, in our case a pheromone, and the other diploid will be able to detect it by a receptor on its surface, and react to it by producing an output - another pheromone. The last diploid in the chain will detect the pheromone and it will in our case cause agglutination of the cells.'''
  
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1: A few years ago.
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'''So how we want to do it?'''
  
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2: Yeah, it’s a pathway and there is some receptor in it.
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'''We want diploids to communicate through pheromones and receptors and that is why they have to have the yeast pheromone mating pathway activated. We also have our own receptors and our own pheromones. More specifically, we use pheromones and receptors from different genera of yeasts. Thus when a diploid detects pheromones from another diploid, production of a different pheromone starts. The yeast cell also produces antibodies on its surface using yeast display. Yeasts naturally have agglutination proteins present on their surface that are used for mating and we are taking advantage of those proteins and adding on top of them the variable regions of our antibodies.'''
  
 +
'''With the antibodies we come closer to the fields of your expertise, because cancer cells or diseased cells have special markers on their surface and these can be recognized by the antibodies that we are displaying on the surface of the yeast.'''
  
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I can explain it. Each haploid cell has this pathway active and has receptors on its surface. When the receptor is occupied by the pheromone of the opposite mating type, the signal comes from the surface to the nucleus and causes expression of mating genes and changes the shape of the cell. When the yeast is in the diploid form, this pathway is inactive, since the diploid cannot mate anymore. So we want to activate parts of this pathway and make the diploid cells communicate with each other but not mate. So we turn off some genes that cause the mating but we turn on the genes that transfer the signal from the surface to the nucleus.  
+
'''Let us simplify it and say that the diseased cell has three different surface markers. So the first diploid is displaying an antibody against one of the markers. When it recognizes this marker on the cell surface, it starts to produce the pheromone. The second diploid recognizes another marker thanks to another antibody and when it detects the pheromone from the first diploid, it also starts to produce another pheromone. Then there comes the last one, it recognizes another marker and when there is a signal from the second diploid, it causes agglutination of cells. To sum up, you could see with your naked eye that there is some reaction. So that is how we want to diagnose a special type of cancer using CTCs in blood. If you know the combination of the markers on the surface, you add our yeasts into a blood sample, wait for two to three hours and determine whether cancer cells of this type are present in the blood or not.'''
  
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We also have our own receptors and our own pheromones. More specifically, we use pheromones and receptors from different genera of yeast. Then when the diploid detects pheromones from the other diploid, production of another pheromone starts.
+
'''The markers we would like to detect are not only for different types of cancer but also for other properties, such as markers designating the cells' origin or the place they are targeting. So they are not only for what tissue the CTC comes from but also for other information that could be find using this approach.'''
  
−
It also produces antibodies on its surface using yeast display. Yeasts naturally have some proteins present on the surface used for mating and we are taking advantage of those proteins and add on top of them the variable region of our antibodies that can detect some markers.  
+
'''The advantage of our project is that if you have the library of alpha-s with different antibodies, and different receptors in a-s. You can connect a different receptor and a different antibody and make your own combination. The vision is that the scientists will have this library somewhere in the freezer and when they need a specific combination, they just mate the yeasts and use them to detect the exact kind of cancer.'''
  
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With the antibodies we come closer to the fields of your expertise, because cancer cells or diseased cells have special markers on their surface, and these can be recognized by the antibodies that we are displaying on the surface of the yeast.
+
<html>
 +
</div>
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</html>
  
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Let us simplify it and say that the diseased cell has three different surface markers. So the first diploid is displaying an antibody against one of the markers. When it recognizes this marker on the cell surface, it starts to produce the pheromone. The second diploid recognizes another marker thanks to another antibody and when it detects the pheromone from the first diploid, it also starts to produce another pheromone. Then there comes the last one, it recognizes another marker and when there is a signal from the second diploid, it causes agglutination of cells. So basically, you could see with your naked eye that there is some reaction. So that is how we want to diagnose a special type of cancer using CTCs in blood. If you know the combination of the marker on the surface, you basically add our yeasts into a blood sample, wait for two-three hours, and determine whether cancer cells of this type are present in blood or not.
 
  
 +
P: If you want we can design an experiment. You will so-called spike the blood by a cancer cell line with no markers, and you can test the concentrations.
  
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1: That is nice.
 
  
 +
O: We can give you vials where there will be cancer cells and no cancer cells and we will not tell you which is which.
 +
  
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The markers we would like to detect are not only for different types of cancer but also other properties. For example markers designating the cells' origin or the place they are targeting. So it is not only for what tissue the CTC comes from but also for other information that could be find using this approach.
+
P: You could try some concentration and determine how sensitive your system is.  
  
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The advantage of our project is that if you have the library of alphas with different antibodies, and in as you have different receptors. You can connect different receptor and different antibody and make your own combination. The vision is that the scientists will have this library somewhere in the freezer and when they need the combination, they just mate the yeasts and use them to detect some exact kind of cancer.
 
  
 +
'''That is very kind of you. It would be very nice for future applications, because we want to focus on this project even after the competition.'''
  
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1: If you want we can maybe design some experiment. You will so called spike the blood by some cancer cell line with no markers, and you can test the concentrations.  
+
'''We think that our approach provides easier and quicker diagnosis than the ones used now. This should be able to detect one CTCs in a million of different blood cells.'''
  
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2: we can give you vials where there will be cancer cells and no cancer cells and we won’t tell you which is which.
 
  
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1: You can try some concentration and determine how sensitive it is.  
+
P: One from a million. That makes sense. It can work.  
  
  
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That is very kind of you. That would be very nice for future applications, because we really want to focus on this project even after the competition.
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'''So it can diagnose cancer in an early stage of the disease.'''
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+
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For our project we think that this is easier and quicker diagnosis than the ones used now. And also there are only a few CTCs cells in a blood sample. This should be able to detect one cell from a million that is actually the cancer cell.
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+
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1: One from a million. That makes sense. It can work.
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+
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So it can diagnose that cancer in an early stage of the disease.
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2: Seems interesting.
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O: Seems interesting.
  
  
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1: I think it’s very good and if it proves it’s really working with the experiment in yeast...
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P: I think it is very good and if it proves it really works with the experiment in yeast...
 +
 
  
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2: I would prefer a color reaction instead of agglutination.
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O: I would prefer a color reaction instead of agglutination.
  
  
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2: I would say so. It’s easier to say okay it’s pink now, then is or isn’t there any precipitation?
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O: I would say yes. It’s easier to say: 'Okay, it’s pink now', then: 'Is or is not there any precipitation'?
  
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1: are you going to detect it in tubes?
 
  
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P: Are you going to detect it in tubes?
  
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Yes, we want to use tubes
 
  
 +
'''Yes, we want to use tubes.'''
  
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1: For example test for blood groups is very nicely visible on the slide because it´s low volume and you can see how erythrocytes ag'''Bold text'''glutinate. But in a tube it will be much more difficult.
 
  
 +
P: The test for blood groups is very nicely visible on a slide because it is low volume and you can see how erythrocytes agglutinate. However, in a tube it will be much more difficult.
  
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We have something to think about, thank you.
 
  
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'''We have something to think about, thank you.'''
  
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1: And just here, the idea is very nice but it’s still limited to the markers you think the cancer cells have. And there may be a problem that some cancer cells when they’re leaving the tumor (undergoing EMT) they can lose their epithelial markers and express some mezenchymal ones.
 
  
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P: And just here, the idea is very nice but it is still limited to the markers you think the cancer cells have. And there may be a problem that some cancer cells lose their epithelial markers and express some mesenchymal (undergo EMT) ones when they are leaving the tumor.
  
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'''When this transition happens how fast is it?'''
 
  
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'''When this transition happens, how fast is it?'''
  
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1: it’s still a question. Because almost all of methods which are used for detection of CTCs are based on the epithelial markers, so we actually have no idea how many mesenchymal-like cells are there. So for example our method is trying to find also those cells which underwent EMT, but the problem with mesenchymal transition is that many of the mesenchymal markers are expressed on white blood cells as well.
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P: It is still a question. Because almost all of the methods, which are used for detection of CTCs are based on epithelial markers, we actually have no idea how many mesenchymal-like cells are there. So for example our method is trying to find also the cells which underwent EMT, but the problem with mesenchymal transition is that many of the mesenchymal markers are also expressed on white blood cells.
  
  
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1: Yes.  
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P: Yes.  
  
  
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1: We would probably do it by some positive and negative selection. So you would not include only positive markers but also markers which we suppose tumor cells cannot have.
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P: We would probably do it by some positive and negative selection. So you would not include only positive markers but also markers which we suppose tumor cells cannot have.
  
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'''
 
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Can you please tell us what the current superior diagnostic approaches are? Do you see the innovation in our approach?'''
 
  
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'''Can you please tell us what the current superior diagnostic approaches are? Do you see innovation in our approach?'''
  
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1: So currently I think the normal diagnosis is based mostly on trial and error. The diagnosis is usually fine, you know there is some tumor, but to choose the correct treatment is difficult because you don’t have online monitoring of the disease. You can do some CT scans or MRIs every half a year but in some cases of aggressive tutors it’s not enough. There is an idea, that CTCs will be used every month or two months to see if there are any changes in the patient. But today there is only one FDA approved test for CTC detection based on the EpCAM molecule, and there are like 20 others which are trying to do it better. I don’t think that any one of them is currently used for routine monitoring of the disease in the Czech Republic. If your project works it might be relatively cheap, which would be a good advantage.
 
  
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P: Currently I think the normal diagnosis is based mostly on trial and error. The diagnosis is usually fine, you know there is some tumor, but to choose the correct treatment is difficult because you do not have online monitoring of the disease. You can do some CT scans or MRIs every half a year but in some cases of aggressive tumors it is not enough. There is an idea that CTCs will be used every month or two months to see if there are any changes in the patient. Today, there is only one FDA-approved test for CTC detection based on the EpCAM molecule and there are about 20 others, which are trying to do it better. I do not think that any one of them is currently used for routine monitoring of the disease in the Czech Republic. If your project works it might be relatively cheap, which would be a good advantage.
  
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'''You said that the CTCs could be used a patient monthly for example. What exactly would you monitor except of the number of the cells?'''
 
  
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'''You said that the CTCs could be monitored in a patient monthly. What exactly would you monitor except for the number of the cells?'''
  
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2: If you have several assays, you can monitor if there are changes in expression of different markers which could also be important.
 
  
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1: For example in our project, we’re trying to monitor the number and then characterize them by single cell sequencing to find out if they are somehow changing the properties of their genome. It brings information whether these cells are just dying cells from metastasis or if they’re alive and evolving to some more aggressive form of cancer.  
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O: If you have several assays you can monitor changes in expression of different markers which are potentially important.
  
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1: Do you want to use it for patients who already have cancer or as a monitoring?
 
  
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P: For example in our project, we are trying to monitor the number and then characterize them by single cell sequencing to find out if they are somehow changing properties of their genome. It brings us information about whether these cells are just dying cells from metastasis or if they are alive and evolving to some more aggressive form of cancer.
  
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The system we’re developing could be used for both applications. At the present stage, we can still choose between both of those possibilities.
 
  
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P: Do you want to use it for patients who already have cancer or as a monitoring tool?
  
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1: Because for example in colorectal cancer, there are some decals occult blood tests which are done every two years after 50 years of age. It is working but it’s not the best examination we can have. If it would be possible to test from blood using your system, that would be great.  
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 +
'''The system we are developing could be used for both applications. At the present stage, we can still choose between both of those possibilities.'''
 +
 
 +
 
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P: For example in colorectal cancer, there are some fecal occult blood tests, which are done every two years after 50 years of age. They are working, but it is not the best examination we could have. If it would be possible to test from blood using your system, that would be great.  
  
  
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2: I don’t think so. The legal problem would be to get it into a clinical environment. If you really want to use it for diagnoses it has to go through all the evaluation processes. But otherwise I don’t think there is problem.
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O: I don’t think so. The legal problem would be to get it into a clinical environment. If you really want to use it for diagnoses it has to go through all the evaluation processes. But otherwise I don’t think there is a problem.
  
  
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2: Each one of them is different.
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O: Each one of them is different.
  
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1: To distinguish between the cells which are released passively from the tumor and those which actively migrate from the tumor is probably the biggest problem.  
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P: To distinguish between the cells which are released passively from the tumor and those which actively migrate from the tumor is probably the biggest problem.  
  
  
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1: It’s not about the diagnosis but about the evaluation of the aggressively of the tumor. Both of them have the disadvantage that you get only a small part of the tumor. In the biopsy you can get a lot of stromal cells and just a few cancer cells depending on where you take the sample from. It’s similar with CTCs, you could only detect the cells which are not important for the future spreading of the tumor. And the question is still how to choose the important ones.
+
P: It’s not about the diagnosis but about the evaluation of the aggressivity of the tumor. Both of them have the disadvantage that you get only a small part of the tumor. In the biopsy, you can get a lot of stromal cells and just a few cancer cells depending on where you take the sample from. It is similar with CTCs, you could only detect the cells which are not important for the future spreading of the tumor. And the question is still how to choose the important ones.
  
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2: There is one other advantage of CTCs: If you’re taking blood it’s not very invasive. If you take the biopsy, you can actually release some cells into the blood and help the tumor to spread. In case of CTCs, they’re already in the blood and you take just a sample.
 
  
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1: And probably those which are really active can survive longer in the blood, so there is chance that when using CTCs you can get more often the cells important for the spreading.
+
O: There is one other advantage of CTCs: if you are taking blood it’s not very invasive. If you take the biopsy, you can actually release some cells into the blood and help the tumor to spread. In case of CTCs, they are already in the blood and you just take a sample.
 +
 
 +
 
 +
P: And those, which are really active can probably survive longer in the blood, so there is a greater chance that when using CTCs you can detect the cells important for the spreading.
 
   
 
   
  
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1: I know there are troubles with diagnosing brain tumors. It is still a question how to find brain tumor cells in the blood. They are there but again, they have different markers. Also some pancreatic tumors, because they are usually detected in a very late stage, when it is too difficult to help the patients.  
+
P: I know there are troubles with diagnosing brain tumors. It is still a question how to find brain tumor cells in the blood. They are there, but again, they have different markers. Also some pancreatic tumors are difficult to diagnose because they are usually detected in a very late stage, when it is too difficult to help the patients.  
  
  
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1: I will start with colorectal cancer again. There are different types of blood tests. And their sensitivity is very high (over 95%). Specificity is a little bit lower, but if you have a positive test, you undergo colonoscopy, which is 100% specific and the sensitivity is also very high. For the imaging methods, it depends on the size. Usually some imaging, like a CT or MRI is the consequence step after a colonoscopy or a positive stool blood test. For CTCs it’s around 80%.
+
P: I will start with colorectal cancer again. There are different types of blood tests. And their sensitivity is very high (over 95%). Specificity is a little bit lower, but if you have a positive test, you undergo colonoscopy, which is 100% specific and the sensitivity is also very high. For the imaging methods, it depends on the size of the tumor. Usually, imaging techniques like a CT or MRI are the consequence step after a colonoscopy or a positive stool blood test. For CTC diagnosis, both are around 80%.
−
 
+
  
 +
= Acknowledgement =
  
 +
* Biomedical Center of Faculty of Medicine in Pilsen
 +
* Mgr. Pavel Pitule Ph.D.
 +
* Mgr. Pavel Ostašov Ph.D.
  
 
{{:Team:Czech_Republic/Template:Bottom}}
 
{{:Team:Czech_Republic/Template:Bottom}}

Latest revision as of 09:45, 12 October 2015

Interview

Motivation

Interview

To get a professional opinion on our project and to learn more about cancer and CTCs, we conducted an interview with two cancer specialists. After the introduction of our project idea, we discussed some particularly interesting points. We received recommendations that helped us to improve our project. This interview was not only helpful and enriching but also led to an agreement to collaborate with the Laboratory of Tumor Biology in the near future.

Key outputs

A suggestion to use a color change instead of the agglutination as an indicator of CTCs' presence. We modified our experiments in module 2 and used GFP as indicator of successful communication between IODs. We are definitely going to extend the use of color indication in further research.

A recommendations to test the agglutination on a slide. We have the possibility to perform our experiments in microfluidics. We appreciated this advice and used microfluidics in many of our agglutination experiments.

The experts

Mgr. Pavel Pitule Ph.D.

Interview

Mgr. Pavel Pitule, Ph.D. is a junior researcher at the Biomedical Center at the Faculty of Medicine in Pilsen. He is the research group leader of the Laboratory of Tumor Biology.

Mgr. Pavel Ostašov Ph.D.

Interview

Mgr. Pavel Ostašov, Ph.D. is a researcher at the Biomedical Center at the Faculty of Medicine in Pilsen. He is a member of the Laboratory of Tumor Biology.

Interview

Could you introduce yourselves and explain your research to us?


P: My name is Pavel Pitule, and I work in the biomedical center of the Faculty of Medicine in Pilsen. Our group is focused on cancer biology. We are looking for biomarkers to use in prognosis and prediction of colorectal cancer. There are two branches of our research, the first one uses molecular methods such as quantitative PCR and microarray sequencing to find mutations or changes in expression in tumor tissues and the second is focused on circulating tumor cells (CTCs) and we are trying to find specific cells released from the tumor into blood stream and characterize them with regards to disease progression.


O: I’m Pavel Ostašov, and I work in the same group. I am mostly in charge of microscopy and processing of sequencing data.


Have you encountered any synthetic biology concepts or applications in your research? What are some examples?


P: I have only heard a little about synthetic biology and have not used it. From what I have heard it can be used for analyzing blood particles.


O: I know there are certain modified cells that release a toxic protein that is able to find the cancer cell. The release of the toxic protein can kill the cancer cell.


What is your view on synthetic biology in research and in real life applications? In your opinion, would the research benefit from using synthetic biology more?


O: It would be nice to use it for detection of changes in cytokines and differences in levels of screening molecules.


P: It could be used for detection of metabolic products of a drug. Some cancer cells can modify prodrugs, which are used in chemotherapy and then expel it outside of the cell. So monitoring cells' resistance to the therapy would be great.


Based on what you’ve said, I think you will like our project.

And since it touches the field of your expertise, we would like to hear your opinion and view on it.

Click here to read how we described our project...


P: If you want we can design an experiment. You will so-called spike the blood by a cancer cell line with no markers, and you can test the concentrations.


O: We can give you vials where there will be cancer cells and no cancer cells and we will not tell you which is which.


P: You could try some concentration and determine how sensitive your system is.


That is very kind of you. It would be very nice for future applications, because we want to focus on this project even after the competition.

We think that our approach provides easier and quicker diagnosis than the ones used now. This should be able to detect one CTCs in a million of different blood cells.


P: One from a million. That makes sense. It can work.


So it can diagnose cancer in an early stage of the disease.


O: Seems interesting.


What is your opinion? Do you see any factual flaws or advantages of this idea?


P: I think it is very good and if it proves it really works with the experiment in yeast...


O: I would prefer a color reaction instead of agglutination.


Why would you prefer a color reaction? To make it more obvious?


O: I would say yes. It’s easier to say: 'Okay, it’s pink now', then: 'Is or is not there any precipitation'?


P: Are you going to detect it in tubes?


Yes, we want to use tubes.


P: The test for blood groups is very nicely visible on a slide because it is low volume and you can see how erythrocytes agglutinate. However, in a tube it will be much more difficult.


We have something to think about, thank you.


P: And just here, the idea is very nice but it is still limited to the markers you think the cancer cells have. And there may be a problem that some cancer cells lose their epithelial markers and express some mesenchymal (undergo EMT) ones when they are leaving the tumor.


When this transition happens, how fast is it?


P: It is still a question. Because almost all of the methods, which are used for detection of CTCs are based on epithelial markers, we actually have no idea how many mesenchymal-like cells are there. So for example our method is trying to find also the cells which underwent EMT, but the problem with mesenchymal transition is that many of the mesenchymal markers are also expressed on white blood cells.


So if you knew what combination of markers to look for then you could find them?


P: Yes.


Could you then distinguish mesenchymal cells from white blood cells?


P: We would probably do it by some positive and negative selection. So you would not include only positive markers but also markers which we suppose tumor cells cannot have.


Can you please tell us what the current superior diagnostic approaches are? Do you see innovation in our approach?


P: Currently I think the normal diagnosis is based mostly on trial and error. The diagnosis is usually fine, you know there is some tumor, but to choose the correct treatment is difficult because you do not have online monitoring of the disease. You can do some CT scans or MRIs every half a year but in some cases of aggressive tumors it is not enough. There is an idea that CTCs will be used every month or two months to see if there are any changes in the patient. Today, there is only one FDA-approved test for CTC detection based on the EpCAM molecule and there are about 20 others, which are trying to do it better. I do not think that any one of them is currently used for routine monitoring of the disease in the Czech Republic. If your project works it might be relatively cheap, which would be a good advantage.


You said that the CTCs could be monitored in a patient monthly. What exactly would you monitor except for the number of the cells?


O: If you have several assays you can monitor changes in expression of different markers which are potentially important.


P: For example in our project, we are trying to monitor the number and then characterize them by single cell sequencing to find out if they are somehow changing properties of their genome. It brings us information about whether these cells are just dying cells from metastasis or if they are alive and evolving to some more aggressive form of cancer.


P: Do you want to use it for patients who already have cancer or as a monitoring tool?


The system we are developing could be used for both applications. At the present stage, we can still choose between both of those possibilities.


P: For example in colorectal cancer, there are some fecal occult blood tests, which are done every two years after 50 years of age. They are working, but it is not the best examination we could have. If it would be possible to test from blood using your system, that would be great.


Do you recognize any legal problems with developing new diagnostic technologies using yeasts?


O: I don’t think so. The legal problem would be to get it into a clinical environment. If you really want to use it for diagnoses it has to go through all the evaluation processes. But otherwise I don’t think there is a problem.


Could you elaborate more on the difficulties of using CTCs for diagnosis? You said that their low number is a disadvantage for diagnosis. Are there any other?


O: Each one of them is different.


P: To distinguish between the cells which are released passively from the tumor and those which actively migrate from the tumor is probably the biggest problem.


If you would compare diagnosing the cancer form CTCs to diagnosis from biopsies. How would you describe it?


P: It’s not about the diagnosis but about the evaluation of the aggressivity of the tumor. Both of them have the disadvantage that you get only a small part of the tumor. In the biopsy, you can get a lot of stromal cells and just a few cancer cells depending on where you take the sample from. It is similar with CTCs, you could only detect the cells which are not important for the future spreading of the tumor. And the question is still how to choose the important ones.


O: There is one other advantage of CTCs: if you are taking blood it’s not very invasive. If you take the biopsy, you can actually release some cells into the blood and help the tumor to spread. In case of CTCs, they are already in the blood and you just take a sample.


P: And those, which are really active can probably survive longer in the blood, so there is a greater chance that when using CTCs you can detect the cells important for the spreading.


Is there any kind of cancer that is harder to diagnose?


P: I know there are troubles with diagnosing brain tumors. It is still a question how to find brain tumor cells in the blood. They are there, but again, they have different markers. Also some pancreatic tumors are difficult to diagnose because they are usually detected in a very late stage, when it is too difficult to help the patients.


What is the sensitivity and specificity of the current diagnostic tests and approaches?


P: I will start with colorectal cancer again. There are different types of blood tests. And their sensitivity is very high (over 95%). Specificity is a little bit lower, but if you have a positive test, you undergo colonoscopy, which is 100% specific and the sensitivity is also very high. For the imaging methods, it depends on the size of the tumor. Usually, imaging techniques like a CT or MRI are the consequence step after a colonoscopy or a positive stool blood test. For CTC diagnosis, both are around 80%.

Acknowledgement

  • Biomedical Center of Faculty of Medicine in Pilsen
  • Mgr. Pavel Pitule Ph.D.
  • Mgr. Pavel Ostašov Ph.D.