November 16, 2021

Detecting sepsis: the role of single-cell

By Conor McKechnie and Dodi Axelson

Detecting sepsis: the role of single-cell

Single-cell sequencing is technology that is giving us new genomic capabilities. Dr. Luciano Martelotto joins us to explain how single-cell sequencing allows scientists to understand cells as building blocks, much like LEGO™, which form part of a much bigger structure such as an organ, a tissue, a disease, and so forth. Dr. James McLaren utilizes this technology to look at septicemia; in his work he is using single-cell analysis to better understand sepsis and to develop a rapid diagnostic test. Single-cell sequencing could hold the key to understanding why the body reacts to infections, and overall to help us advance healthcare.

Join Dodi, Conor and their guests, Dr. Luciano Martelotto, Scientific Director of Single Cell Lab at Harvard Medical School, and Dr. James McLaren, Systems Immunity lecturer at Cardiff University, in the latest episode of Discovery Matters.

DODI: We've talked on previous episodes of Discovery Matters about all kinds of cells, haven't we, Conor?

CONOR: Yes, they have been called T-cell therapy and CHO cells and 'she sells seashells by the seashore' cells.

DODI: But today let's look into the world of single cells, which kind of touches upon so many other topics we've discovered on this podcast.

CONOR: So, is this like a Tinder™ episode on cells, their trying to match up? What specifically about single cells should we be looking at?

DODI: No, today let's look at single-cell sequencing, how it is advancing our understanding of biology and how it can be applied to improve diagnostic tests. So, that is what matters on today's episode of Discovery Matters. Luciano Martelotto is the Scientific Director of Single Cell Lab at Harvard Medical School. To understand how Luciano ended up in Harvard, we must go back a few decades to a small town in Argentina.

LUCIANO MARTELOTTO: When I was a kid, I always wanted to be a vet, until someone...

DODI: ...a kid from his high school...

LUCIANO MARTELOTTO: ...told me about DNA. And I just got struck by lightning! And I thought, 'Okay, that's exactly what I want to do for the rest of my life. And I want to understand what it is'.

DODI: And that was the start of Luciano's career in biotechnology.

LUCIANO MARTELOTTO: ...Which at the time in Argentina was actually just starting, and I thought it was amazing. It was very difficult because I was coming from a very small town in which things were very town-ish, very simple. So, I had to learn a lot of things, and from scratch understanding what it was to live in a big city because I had to move to a big city. And also, I needed to learn things that I hadn't learned at school, like very basic stuff from math to biology. And I thought 'Okay, this is challenging. But this is exactly what I want to do.' And challenge was the fuel, essentially, of my career and just finding difficult things and trying to understand how those work.

DODI: Now this career of Luciano's so far has been a whirlwind.

LUCIANO MARTELOTTO: So, I first worked a little bit in bacteria, and then I moved to plant genetics for my bachelor’s degree. And while I was doing my PhD, a guy from Australia, went to Argentina and said 'Look, would you like to go to Australia to work with us?' And I said, 'Yeah, why not?'

DODI: And this kid from a small town in Argentina suddenly found himself in Australia as soon as he finished his PhD.

LUCIANO MARTELOTTO: Essentially, the idea of that was just to improve some crops, and it was a pretty successful year with patents and stuff like that, but I wasn't really feeling it. It's not what I really wanted to do for the rest of my life, I wanted to do something more human-related. I had completely abandoned the idea of working with animals.

DODI: So, he managed to contact someone at the Johns Hopkins Institute in the US.

LUCIANO MARTELOTTO: And he took me in! As you know, I was a plant geneticist, so a molecular biologist in plants. And the guy said, 'Look, I, I know that you can do this'. And so, I had to start reading about cancer again, that was my challenge.

CONOR: So, he's essentially kind of starting all over again, from scratch?

DODI: Kind of. His focus now is on cancer. And once, he took a book out of the library called ‘Biology of Cancer’ by Robert Weinberg.

LUCIANO MARTELOTTO: I read it five times from page one to the end. I got that all in my head. It was amazing to see all these new things that were totally different to plants. And that's how I got my love for the human biology in general.

DODI: This shifted his focus to human genomics, then he started thinking back to his time in Australia.

LUCIANO MARTELOTTO: While I was doing my postdoc in Australia, we were sequencing whole genomes of xenograft animals. There are mice that we have injected or introduced some human cells into to generate tumors in these animals. But we didn't have any understanding of what we were doing in terms of the informatics, so in terms of how to interpret the data. So, I always thought that if I ever had the chance to investigate or to do more on that, I would do it. That's when I learned about single-cell genomics. That's when I fell in love with the single-cell world.

DODI: But his journey was not over.

LUCIANO MARTELOTTO: I came to New York for a conference last year, then I happen to get in touch with someone here from Harvard that said, 'Look, we need a person just like you here. Do you want to come?' And I said, 'Okay, well, I need to ask my wife!' So, after difficult negotiations, we ended up in Boston and that's where I am now.

CONOR: Wow, what a journey! So, what exactly does Luciano do in Boston at Harvard Medical School?

DODI: Today, the chameleon that is Luciano Martelotto is all about single-cell sequencing. Now before we get into the nitty gritty of single cells, it's important to understand that Luciano is a realist.

CONOR: Okay, so in what way?

DODI: So oddly, he spends a lot of time telling people not to conduct single cell experiments.

LUCIANO MARTELOTTO: But not because I don't want them to do single-cell sequencing. The problem is that people think that because the technology is available, everybody's ready for the technology. And that's not true. The fact that there are some protocols available and are optimized, that doesn't mean that the user is optimized for the protocol. So, I always insist, if you have the opportunity to save money, just save the money for the rainy day! You don't have to do crazy experiments if they are not needed. The fact that the technology is there doesn't mean that you can use it or that it is necessary. It is like saying, 'Oh, because now we can see from far with the Hubble to see stars that are light years away, we're going to use the Hubble to see how the moon looks like.' You have to use it for things that are absolutely necessary. But not everybody understands that.

CONOR: Okay, so not everybody should be doing it for its own sake. But just help me here. What is single-cell sequencing apart from something difficult to say six times fast?

DODI: Well, imagine you have a pile of LEGO™ in front of you.

CONOR: Okay, so I'm a bit weird about my LEGO™. I like to separate them out with my reds, and my yellows, and my greens, and my blues, and my transparents, and then I like to separate them into like, never mind, okay I can imagine it.

DODI: Exactly! Just watch your feet, because we all know how much it hurts when you step on those bricks.

CONOR: Great parent traps, they were the best. So, where were we… LEGO™? What's going on?

DODI: Yeah, exactly.

LUCIANO MARTELOTTO: Okay, so we have a little piece of LEGO™. And for a non-expert eye, all of the pieces look the same. But for those that are expert, every single piece is different. So, you have one that has pieces that are larger to stick them together, some of those are round, and there'll be some longer and a little bit thinner. That's what we develop the different methods, all the research, and development. The different methods are used to be able to get from every single piece of LEGO™ as much information as we can. So, now we can separate them much better or group them into things that are the same and differentiate things that are slightly different. And then based on that knowledge from the individual pieces, we can start saying 'Okay, well, this piece looks like this one, but it should not go here. It should go here because it makes sense to fit it with this one'. And so on, so forth.

CONOR: Okay, so it's all about getting as much information from all the little pieces of LEGO™ so we can separate and group them out from here into my little boxes of you know...

DODI: ...of good colors. And basically yes, and Luciano says this relates back to human atlases.

CONOR: So, we did a human atlas thing, right the way back when we were like at the very beginning of the podcast series.

DODI: Yeah! We encourage you to dip back into the archives and listen to that. That was a fun interview with Mathias Uhlén. And in fact, Luciano says the Human Cell Atlas is basically cataloguing those LEGO™ pieces.

LUCIANO MARTELOTTO: It is just getting as much information as it can from every single one of the pieces. And all these pieces form part of a much bigger structure, which could be an organ, it could be a tissue, it could be a disease, and that it's what you end up building with the LEGO™. So, you know, it could be a castle, it could be a car, it could be anything.

CONOR: Okay, I really like this analogy. It's really easy to grasp. So, practically, how is single-cell sequencing and analysis – got my tongue around that one – how is that used in the real world?

DODI: To answer that, we have to meet James.

JAMES MCLAREN: So, I'm currently a lecturer at Cardiff University. I'm also a Lab Head.

DODI: And he focuses his research on sepsis.

JAMES MCLAREN: Sepsis is essentially something that's not well known about. A lot of people have heard of the term septicemia, which is something called blood poisoning. But actually, sepsis is when your immune system overreacts to infection, so everything goes a bit haywire and out of control. We don't know the full reasons why the tests involved at detecting it aren't as fast as we'd like. So, a lot of people are now kind of using what we can to try and learn more, and to try and detect it faster, and to try and find new therapies that treat it. Sepsis is one of the biggest killers globally. I think one in five people globally die of sepsis or complications linked to sepsis. And that doesn't always occur during the early stages. Sometimes it can occur months or years afterwards, when perhaps the immune system hasn't recovered and they're more susceptible for other infections coming along later on.

CONOR: So, what's the situation with diagnosis today? How does that work?

JAMES MCLAREN: There's a number of clinical tests that are used to diagnose it at the bedside. Beyond that, one of the gold standard tests, as we call it, is to take a blood sample, and to culture just to see if there's any bacteria in the blood. So, the majority of infections that are called sepsis are bacterial but is not always the case. With sepsis, you imagine it's a massive cascade that the faster you detect it, the better your chances are at surviving it. So, if you are diagnosed very late the chances of surviving it are much lower. So, what me and others are trying to do is to find ways to diagnose it faster, learn more about what's happening to the cells, when this immune system goes out of control, and to see then if we can develop things that improve patient outcome when sepsis happens.

CONOR: So, James wants to understand more about what's happening at the cellular level, how's he going to do this?

DODI: So recently, James has been using single-cell analysis in his work to better understand sepsis and develop a rapid diagnostic test.

JAMES MCLAREN: The immune response to infection is very complex. It has lots of different types of cells involved. So, what single-cell analysis enables us to do is to be able to look at lots of different cell types at a single-cell level. So, we can identify specific changes in small groups of cells, if not individual cells, that are driving huge changes in body. This is something that we weren't able to do 20 years ago, it's just expanding our knowledge of what is going on in any person who's going through sepsis.

DODI: Time is so important here!

JAMES MCLAREN: What happens at 24 hours post-diagnosis will be very different than, say, two hours post-diagnosis. So, it's really critical that we try to learn more at the early stages.

DODI: For James, single-cell analysis is less like LEGO™ and more like trying to do an experiment on a pool full of fish. Now that sounds slippery!

JAMES MCLAREN: You take each fish and put them in their own little pool. You then give them, let's say, a new bit of food or a treatment. Instead of that treatment going to one pool across all the fish, each fish gets their own individual treatment. And then you can examine the differences that happen across a certain individual. It's been able to look at what we call the transcriptome, which is a fancy word for looking at how genes change at transcriptional level on a global scale. So, it's been able to do this at a single-cell level so we can tell the difference of what's going on in one cell from another even when a very similar type.

CONOR: So basically, hyper targeting to find the information that you actually need.

DODI: Spot on.

CONOR: So, it seems that single-cell analysis is technically still relatively new as a technology. What are the technical challenges and barriers here?

JAMES MCLAREN: When we use it, the level of sequencing is not as deep as it would be if you looked in a group of cells together. So, we turn to something called read depth, and this read depth could be better. And if it was better, then I think it would be the kind of route that most people would go. Things are getting a lot better; I mean it's advanced so much since it first came out. More and more people use it, and costs are coming down. It's more widely used. So, like any of these new techniques that come along, it doesn't take long before things become a lot easier to use and better and then becomes the sort of technique that everyone is using.

CONOR: And so, what does James see as the next step, the future of single-cell analysis and sequencing?

DODI: He talks about a kind of harmonization.

JAMES MCLAREN: I feel given the advancements that are being made in lots of different sequencing technologies that they will all become quite harmonized together. The kind of idea that you could do a single analysis using almost what people call 'a lab on a chip'.

CONOR: Okay, so we are getting to lab on a chip, right?

DODI: Right. Basically, what it sounds like. A tiny device that integrates and automates multiple lab techniques into a system that fits on a chip.

CONOR: That's fantastic. It's not like a chip that you eat with mayonnaise in Holland or a chip that you eat with beer in Texas. It's a proper chip. So, people have been talking about this for ages, and now it's actually happening.

JAMES MCLAREN: I think that's where I see it going. Huge advancements have been made in recent years, and it's making it more accessible for all and will change the game.

CONOR: So, if we stick with the future for a while, how does Luciano in Harvard see the application of single-cell sequencing impacting health and healthcare if he looks ten years ahead?

LUCIANO MARTELOTTO: I think that single-cell sequencing is just an intermediate form of what is coming in the future. I think people are using it right now because it's the newest and most influential technology in terms of how new technologies are evolving, and also influential in the way that things are studied. But we are seeing more and more that spatial analysis is taking off.

CONOR: Okay, spatial analysis, we've heard of that. It's basically looking at cells in the context of the original tissues that they're in and studying the cells where they actually come from.

DODI: Right.

LUCIANO MARTELOTTO: I think that in the future – and I don't know how far in the future but is probably very, very close – we won't even need to take the sample out of the patient to do this type of analysis. I mean, it sounds science fiction, okay. And it sounds like something that you will see in an Avatar movie. But if you think about five years ago, we would not have even considered the possibility of doing spatial transcriptomics in the way that we are now. We are heading towards something that is going to come in the next 10 years that I cannot even imagine because I know the new tech can bring new tech. And this is what is happening now.

CONOR: I love the way new technology means new technology means new capabilities. It's just glorious.

DODI: It's one of those virtuous circles of science.

CONOR: We've got the virtuous circle of science of single-cell sequencing.

DODI: Say that six times faster.

CONOR: Okay, single-cell sequencing, single-cell sequencing, single-cell sequencing, single-cell sequencing. I can say that!

DODI: And on that medley, we say thank you for listening to this episode of Discovery Matters. The executive producer of Discovery Matters is Andrea Kilin and was produced with the help of Bethany Grace Armitt-Brewster. Editing, mixing and music by Tom Henley and Banda Produktions.

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