We cross the pond for this episode, which focuses on CAR-T cell therapy. Join us to meet Raquel Munoz and learn about what she thinks will be the future of cancer research.
Blood is a symbol of life, which makes sense given that it plays such an important role in so many body functions, including our immune system. Blood makes up approximately 8% of your normal body weight and unfortunately, cancers of the blood, including lymphoma and leukemia, account for ~10% of all diagnosed cancers in the U.S. each year.
CAR-T cell therapy has emerged as a promising method to engineer a subject’s own immune cells to fight bloodborne cancer. Our guest for this episode, Raquel Munoz from the Hospital Universitario Virgen del Rocío in Seville Spain, is doing research in this exciting CAR-T cell therapy space. Specifically, she is working to develop methods to help better quantify and understand the expansion of CAR-T cells in the body to help monitor treatment and predict outcomes. We learn about why digital PCR was selected for her work and how it’s helped raise confidence in the results they’re getting. We even hear about how she believes this treatment will find success in treating solid tumor cancers.
In Cassie’s career corner, we learn how Raquel found her career path and love of immunology and working in a hospital setting. Raquel also shares some great career advice, stories of lab mishaps, and the dangerous hobby that she says is some of the only time she’s not thinking about work or problems.
Visit the Absolute Gene-ius page to learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.
Cassie McCreary 00:00
I was just thinking, based on that conversation we just had that if I were to donate my body to science, they'd probably just return it. No, thank you.
Jordan Ruggieri 00:10
No thanks.
Cassie McCreary 00:13
We've seen better.
Jordan Ruggieri 00:26
Welcome to Absolute Gene-ius, a podcast series from Thermo Fisher Scientific. I'm Jordan Ruggieri.
Cassie McCreary 00:31
And I'm Cassie McCreary. And today we take Absolute Gene-ius overseas for a fabulous conversation with Dr. Raquel Munoz of Seville, Spain
Jordan Ruggieri 00:40
Raquel recently finished her doctoral studies and works as an immunologist on the forefront of innovative cancer research. She's opened our minds to what the future of cancer research looks like. And we're excited to share our conversation with you today.
Cassie McCreary 00:53
How exciting for us that we've gone international.
Jordan Ruggieri 00:56
I know it's so cool to have our first international guests on the podcast. I think it's absolutely amazing.
Cassie McCreary 01:02
Some might say it's absolutely "Gene-ius."
Jordan Ruggieri 01:07
We're awful. I use absolutely in everything. Now I can’t get away from it.
Cassie McCreary 01:12
Can't stop, won't stop.
Jordan Ruggieri 01:18
Raquel, thank you very much for joining us today on the Absolute Gene-ius podcast. We're really excited to have you with us and to talk about your research.
Raquel Munoz, PhD 01:26
Hi there. Me too. Thank you for inviting me.
Jordan Ruggieri 01:29
Can you give us a background of what your research is and who you are for our listeners?
Raquel Munoz, PhD 01:35
Yeah. My name is Raquel. I work at Hospital Universitario Virgen del Rocío in Seville in Spain. And our research is about CAR-T cell therapy. Have you hear about it?
Jordan Ruggieri 01:51
Yes. Yeah, CAR-T is awesome. Can you talk a little bit about what CAR-T is for the listeners as well.
Raquel Munoz, PhD 01:59
It's a type of immunotherapy. We can use the immune system to protect from an illness. And so, we can modify the immune system and for the treatment of this illness. And for example, with the CAR-T cell therapy, we obtain T-cells that are immune cells, these are the fighters of the immune system. We can modify them in the lab and put our receptor, a specific receptor, that can find and kill cancer cells. So, we create the CAR-T cell that is the crowning of chimeric antigen receptor T-cells, because we have added these chimeric receptors in the cell surface. So, when we create these CAR-T cells, we can reintroduce them into the into a subject and then the CAR-T cells will find and kill the cancer cells in a specific way.
Jordan Ruggieri 03:10
That is, that is incredible. So, when you are working with CAR-T, can you describe a little bit of how you know a modification was made properly?
Raquel Munoz, PhD 03:24
Yeah, we have markers gene. So, we try, we try to amplify these marker genes. So, we know that the CAR-T cells that we have made have a specific gene. So, if we amplify this gene, we know that we have done the CAR-T cell well. And if we don't see any amplification, we have failed with the, with the construction of the CAR-T cell.
Jordan Ruggieri 03:58
Do the CAR-T cells stay a long time in a subject, or do they degrade over time?
Raquel Munoz, PhD 04:07
We are researching about this because it's important to know how many days, or even months, the CAR-T cells is still in and do the work. Because we want that they work only in a period of time. Because if they are still and they still killing, maybe we can have side effects. We don't want to have it. Our main that we want to achieve is to measure how many times these cells is still in the blood. So, we try to measure and describe what is the curve of the expansion of these CAR-T cells.
Jordan Ruggieri 05:05
Very interesting. What are you hoping the ultimate outcome is associated with your work?
Raquel Munoz, PhD 05:14
We want to achieve to monitoring the expansion curve of the CAR-T cells. And until now, we have described three groups. The first group that we have described, it's a group that expands the product that we have infused between day 10 and 11. The second group, it's late expansion group, and they expand the product after day 11. And there is a third group that has no expansion. So, we are trying to compare if there are any difference between these three groups. And we have seen in our preliminary research, that maybe it could be any difference in terms of side effects or failure of the CAR-T cell therapy. So, our mark is to, is trying to prevent and predict the outcome of the therapy.
Jordan Ruggieri 06:35
You talk about expansion. Can you elaborate on what do you mean by expansion? Is that growth of the modified cell line within a subject?
Raquel Munoz, PhD 06:46
Yes. We talk about the increase of the levels of CAR-T cells that we can detect on the blood. Because there is an increase first, and then a decrease. So, we don't know and this is what we want to know, that if this increase and decrease is related to the effects of the CAR-T cell. And if the amount of the CAR-T cell that we can, that we can detect in the blood is related to the effect of the therapy and the side effects also.
Jordan Ruggieri 07:29
Interesting. So, you, you are trying to understand what happens when you put the CAR-T cell, the modified cells into the subject to determine what, you know, the cells grow and then the cells, and reproduce and then the cells decrease and how that impacts the subject and outcomes of that CAR-T therapy? Is that accurate?
Raquel Munoz, PhD 07:59
Yeah. Because maybe if we understand this curve of expansion and maybe we can predict the outcome. And, for example, if we, if we don't see an expansion, maybe we could give a second dose of CAR-T for example. If maybe we see early expansion or have a high amount of CAR-T cells, maybe it could be related to side effects and maybe we could give some drugs to prevent the side effects that could be fatal. For example, we talk about CCT, or cytokine storm. So, if we can prevent these, maybe the outcome it will be better.
Jordan Ruggieri 08:57
Raquel, what technologies do you use in your research? Do you run NGS or digital PCR?
Raquel Munoz, PhD 09:06
Yeah. We use digital PCR in fact. And we tried to measure the expansion of CAR-T cell at first with real time PCR, but we have a lot of problems. Because as you know, with real time PCR we need to amplify a reference target and in the same reaction, we have all the reagents and the primers to amplify our target, but also the target reference, the reference target sorry. And so, in cases of low frequency targets, as our case, there is a high probability of competition between the reagents. And maybe we could amplify only the reference target because there is a high proportion if we compare with our target. So, these problem doesn't exist with digital PCR because thanks to partitioning, we have micro amplifications. So, we do an absolute quantification, we don't need a reference. So, we obtained an absolute quantification. And in our research, this is a great advantage. So, we decide to use digital PCR here.
Jordan Ruggieri 10:35
Awesome. So, digital PCR instead of comparing to a standard curve or to a reference, you're able to take that copies per microliter number and use that to plot your expansion curve? Is that accurate?
Raquel Munoz, PhD 10:54
Yeah, this was the clue of our research, because with real time PCR, in some cases, we didn't, we wasn't able to amplify our target because there was very low proportions.
Jordan Ruggieri 11:13
Oh, I see. So, your limit of detection is much lower when it comes to digital PCR? Your limit of quantification is lower? So, you're able to see those smaller changes easier? Is that accurate?
Raquel Munoz, PhD 11:30
Yeah. And that's a great advantage for us. Because with digital PCR, we are assured that if we don't see amplification, we are in the third group that I am, as I said before. But maybe with real time PCR, we are not sure that if we are in the third group, and there is no amplification, there is no expansion. Or maybe there are competition between the reference and the target. So, using the digital PCR, we are sure about this.
Jordan Ruggieri 12:08
That's great. Do you still use reference genes, when you use digital PCR to quantitate? And do you use a smaller number of them compared to qPCR?
Raquel Munoz, PhD 12:24
Yeah, in digital PCR, we use a reference because we want, we want to know that if we don't see amplification of our target, but we see amplification of the reference target, we know that they are cells, but they are not a CAR-T cells. And also, we use the reference to normalize different measures of different days because maybe in different days or we have different amount of cells. So, we will use it to normalize.
Jordan Ruggieri 13:10
Makes sense. So, you use a reference to make sure that the amplification process is working so if you end up with let's say zero, right, no amplification of the target you're looking for, you know that the process still worked and it's a true zero? And then you also use that reference to normalize and make sure it's an equivalent quantification across multiple days?
Raquel Munoz, PhD 13:42
Yeah, yeah.
Jordan Ruggieri 13:43
We talked a lot about digital PCR and even qPCR. Do you use any other methods? Any cell culturing or next generation sequencing or, what does a day in the lab look like for you?
Raquel Munoz, PhD 13:58
Yeah, it depends on the area. For example, in the histocompatibility, we use a lot of molecular biology like a NGS. In autoimmunity, we use more and cell culture and IIF, it's the acronym of immunofluorescence, indirect immunofluorescence. Yep. And in immunodeficiency, we use a lot of flow cytometry.
Jordan Ruggieri 14:34
Interesting. A lot of different techniques. I actually I forgot a question here. I want to go back a little bit. When you are working with the CAR-T cells and the process, how do you modify those cells? So, do you use CRISPR Cas-9 as part of your modification workflow?
Raquel Munoz, PhD 14:54
Yeah. This part has been made by my colleagues of hematology. I totally, not for me, but yeah, they use these technology. Yeah.
Jordan Ruggieri 15:08
Can you elaborate, or even just from your knowledge, on what that might entail when making modifications to the, to the cells?
Raquel Munoz, PhD 15:17
Yeah. We obtain the T-cells and cultivate them, and we try to expand them in the culture. And then we use this technology that we have talked about, and we put the gene that codes and then the chimeric antigen receptor. So, the T-cell will express this receptor in this cell surface. So, now we have create the CAR-T cell, we expand these CAR-T cells and then we can use them.
Jordan Ruggieri 16:03
That's incredible. Amazing, amazing research there.
Raquel Munoz, PhD 16:07
Yeah. I think it's the future of the cancer treatment.
Jordan Ruggieri 16:13
Are CAR-T therapies more common now? Or are they working only for particular sets of illness?
Raquel Munoz, PhD 16:20
The CAR-T therapy it's more studied in lymphomas and leukemias because the cancer cells are in the blood. But I know that and nowadays there are a lot of research that are trying to use CAR-T cells in solid tumors and the results are good. I am sure that we are going to hear about it more and more.
Cassie McCreary 16:54
Taking a quick break from our conversation to tell you about Applied Biosystems™ QuantStudio™ Absolute Q™ dPCR system. This instrument enables quantification of your targets without the need for standard curves, in only 90 minutes. Digital PCR can be as simple as preparing your samples, loading onto the plate, and running the instrument.
Jordan Ruggieri 17:13
Unlike other digital PCR systems, the Absolute Q dPCR instrument does not use emulsion or other droplet-based methods to compartmentalize reactions. In fact, the microfluidic array plate (MAP) technology enables consistent delivery of more than 20,000 micro-chambers. It's a great solution for anyone looking to quantify gene targets.
Cassie McCreary 17:34
And Thermo Fisher Scientific has a suite of dPCR assays for applications like AAV viral titer quantification, liquid biopsy analysis, and wastewater surveillance. You can learn more at www.thermofisher.com/absoluteq or visit the Absolute Gene-ius webpage. Again, that's www.thermofisher.com/absoluteq or visit the Absolute Gene-ius webpage.
Jordan Ruggieri 18:02
The Applied Biosystems™ QuantStudio™ Absolute Q™ dPCR system is for Research Use Only. Not for use in diagnostic procedures.
Jordan Ruggieri 18:10
Let's get back to our conversation.
Cassie McCreary 18:16
Bienvenidos el Cassie's Career Corner. I don't know how to say corner in Spanish. So, this is the point where I will ask you a little bit about your studies, like academic studies, your education, your career path, a little bit about kind of advice for students and that type of thing. So, can you give us, please, an overview of your academic studies, because you graduated relatively recently.
Raquel Munoz, PhD 18:50
Yeah. I studied biomedicine and then I did a master of clinical diagnosis and lab diagnosis. In my work practice at the master, I realized that I loved hospitals and love hospitals. So, I decided to specialize in immunology. So, I have to, I had to do an exam to do this specialization because it's the system here in Spain. And I passed this,
Cassie McCreary 19:31
Yeah!
Raquel Munoz, PhD 19:34
This exam. And I could choose between different speciality, specialization, and I choose immunology. And I have studied different areas of immunology for four years. And now I'm officially an immunologist.
Cassie McCreary 19:47
Yeah!
Raquel Munoz, PhD 19:48
And I have been working at a hospital as immunologist for two months in Barcelona. And now I'm trying to finish my doctoral thesis.
Cassie McCreary 20:04
So, how did you decide, because at least the way that it would work over here in the U.S., if following a route like yours, a person would probably have to decide between going down PhD studies and research or medical school. Sometimes people do both. And you can do an MD PhD. How did you decide you wanted to go more down the research route versus like medical school, for example?
Raquel Munoz, PhD 20:30
Yeah, I didn't know if I decided.
Cassie McCreary 20:35
Okay.
Raquel Munoz, PhD 20:36
Yeah, I only...
Cassie McCreary 20:37
It's never too late, so.
Raquel Munoz, PhD 20:41
I loved research and know more about human physiology and human illness. So, any project that I could that I could be in, I tried.
Cassie McCreary 20:58
Great! What is it about immunology that you like so much?
Raquel Munoz, PhD 21:01
I think it's an area where you can learn about any illness or any physiology process, because immunology is related to anything. Immunology is related to cardiology, to neurology, and gastrointestinal, nephrology. So, I think it's, it's a complete way of know more about human physiology and illness.
Cassie McCreary 21:41
Yeah, it has a like a wide range. So, it keeps things very interesting, I would imagine, to study, right? Because there's so many different, I guess, applications. For somebody who's maybe just starting out, either in their career. or towards earning a PhD or advanced studies, what is a piece of advice you would give them?
Raquel Munoz, PhD 22:02
Maybe I can say to them that they, “Ask people for their jobs.” Because I am, I think I didn't know very well what people do in their jobs. For example, I didn't know that a doctor could be also immunology. I thought that doctor is only about surgery, or so. I will say to them that they have to ask and make their own opinion about different jobs and in degrees and careers. So, they can make a good decision if they know what people do in their jobs.
Cassie McCreary 22:52
I think it's probably natural for a lot of people at one point or another to feel a little bit of maybe self-doubt during their research, or question if it's something they want to continue. Did you ever experience something like that? And if so, how did you move past that?
Raquel Munoz, PhD 23:11
Yeah, yeah, of course. The research is very hard, because you have many questions and you have to try different things to try to answer these questions. And more, the great majority of these things that you try, they fail. And these are also results, but when you are trying to do things, and you fail, you fail, it's very hard. I think it's frustrating, but when you have a result and you see that all the work that you have done, you could express, and you could write a great article or show your results to your colleagues. And it's, it is also very, very happy for you. So, I think there are good things and not as good things. But the research it's very, it's amazing.
Cassie McCreary 24:29
It sounds like having a very good support system is really important in those times, you know, having colleagues who will support you and other students and your mentor and all that. And I think you hit on an important point, which is I think people forget that a lot of science is built on failure just as much as it is success. People tend to just forget about the failures and move past and not cover that and always glorify only the success, but a lot of times took a big degree of failure to get to that success right?
Raquel Munoz, PhD 25:02
Yeah. I think that to have a positive result, you have to have 100 negative results.
Cassie McCreary 25:12
Right. In your time in the lab, for you, what was your most exciting or proudest moment?
Raquel Munoz, PhD 25:20
I think my proudest moment was the first time that I saw my name in an article, as an author. It was the description of a new allele or something like that. It not. It wasn't a very important article, but it was my first article.
Cassie McCreary 25:40
Yeah!
Raquel Munoz, PhD 25:41
This was, it was amazing.
Cassie McCreary 25:44
Yay. Okay, how about something, you don't have to share it if you don't want to, but how about something that's, that was maybe like a mistake that you made in the lab or something funny or embarrassing that might have happened sometime? Jordan has a good one if you need one, so.
Raquel Munoz, PhD 26:02
I think I have a hundred of these.
Cassie McCreary 26:06
Okay, all right.
Raquel Munoz, PhD 26:07
I have one that is embarrassing but also funny. When I was in my, in my first week, I think yeah, in their lab or in the hospital lab, I was looking for what some samples in the fridge, but a in a room that is fridge. It's a room, but the temperature is under 20 degrees. So, I was looking for my samples and suddenly the door was locked.
Cassie McCreary 26:41
Oh no.
Jordan Ruggieri 26:43
Oh no.
Raquel Munoz, PhD 26:43
And I thought, I thought, "Keep calm and get your phone and phone a colleague and he will open the door." I have no phone.
Cassie McCreary 26:53
Oh no.
Raquel Munoz, PhD 26:54
So, I started shouting out loud. Because I thought I would die. Frozen.
Cassie McCreary 27:02
Oh no.
Raquel Munoz, PhD 27:03
Yeah, I was shouting out loud and for 10 minutes, I think. And then a colleague opened the door and said "Are you crazy? This this red button if you press and if you push the button the door open."
Cassie McCreary 27:25
Like an emergency button.
Raquel Munoz, PhD 27:28
It's a security feature.
Cassie McCreary 27:30
Yeah.
Raquel Munoz, PhD 27:31
It makes a lot of sense because it's a freezer. But I didn't know, and I think all the lab was laughing about me for…
Cassie McCreary 27:41
Oh no.
Raquel Munoz, PhD 27:42
For days. But I was very happy for not dying frozen in my first week.
Cassie McCreary 27:53
Oh, that's a good one.
Jordan Ruggieri 27:57
I don't know if people can, I don't know, understand how many mistakes you make in that first week or so. I made so many funny mistakes my very first week in the lab.
Cassie McCreary 28:11
It's how you learn.
Jordan Ruggieri 28:13
It's how you learn, yeah.
Cassie McCreary 28:17
Because they're never going to cover that in school. You have to mess up that's how you learn. And everybody needs a little bit of a break from work and research and all that. What do you enjoy outside of work?
Raquel Munoz, PhD 28:34
And I enjoy a lot doing pole dance. Do you know what it is?
Cassie McCreary 28:41
Pole dancing?
Raquel Munoz, PhD 28:43
Yeah.
Cassie McCreary 28:46
Raquel, that's awesome! That's great exercise I've heard.
Raquel Munoz, PhD 28:48
Yeah. Yeah. Strong.
Cassie McCreary 28:52
Okay, excellent. That is so good.
Raquel Munoz, PhD 28:55
I think it's and the only hours that I don't think in the job or any problem or because you have to be very concentrate. Because if you are in the pole and you.
Cassie McCreary 29:12
You fall off.
Raquel Munoz, PhD 29:15
Yeah, it's dangerous. It's dangerous.
Cassie McCreary 29:19
I believe it.
Jordan Ruggieri 29:20
My sister does the silk dancing, from the silks that hang from the ceiling.
Cassie McCreary 29:26
Like the aerial acrobatics?
Jordan Ruggieri 29:28
Uh huh. Yeah, that's I don't know how anybody does the poles or the silks I can barely hold myself up for three seconds.
Cassie McCreary 29:45
Oh, that's so funny. So yeah, it's important to take a break and get your mind off of work. Otherwise, you'll run out of ideas. You'll get burned out, right? So, Jordan now that now that's all I can picture is you like just falling off of a pole. Raquel, what would you like to see out of your career in the next maybe five years or ten years?
Raquel Munoz, PhD 30:16
Yeah, it's a great question.
Cassie McCreary 30:19
You don't have to know, it's okay.
Raquel Munoz, PhD 30:22
Yeah, I don't know . The only thing that I want is finish my doctoral thesis and yeah, I wish in five years.
Cassie McCreary 30:36
You will.
Raquel Munoz, PhD 30:36
Yeah.
Cassie McCreary 30:40
You will.
Raquel Munoz, PhD 30:41
Yeah. For my, for my mental health.
Cassie McCreary 30:45
Yes. That's so funny. All right. Great.
Jordan Ruggieri 30:49
Well, Raquel, thank you so much for joining Absolute Gene-ius today. You are our very first international guest, believe it or not. So, we are very excited to have you on and to have you be a part of this. So, thank you very much for your time.
Raquel Munoz, PhD 31:05
Thank you very much. I was very, very confident because of you. Thank you.
Jordan Ruggieri 31:13
That was Dr. Raquel Munoz at Hospital Universitario Virgen del Rocio in Seville, Spain. Thank you so much for joining us on today's episode of Absolute Gene-ius. It was produced by Sarah Briganti, Matt Ferris, and Matthew Stock. Stay curious and we'll see you next time.
Cassie McCreary 31:31
By the power vested in us, we are gene-iuses Yeah, we're going to have to be self-proclaimed because there's not a single institution that would sign off on that statement.
Jordan Ruggieri 31:39
It's the institution of a home printer. That's what it's called.
Cassie McCreary 31:43
Did you know, speaking of home printers, I am ordained?
Jordan Ruggieri 31:47
Oh.
Cassie McCreary 31:47
I should just I should do the same thing but genius.