S2, E07: In Vitro 3D Models: A Blueprint for Drug Development

 

About This Episode

Three-dimensional models are primed to play a vital role in the future of drug development. By recreating tissue that mimics human systems, scientists can monitor disease progression and evaluate the effects of drug candidates. Join Drs. Elizabeth Anderson and Ian Waddell as they explain the evolution of biologic modeling and how 3D models are being used to develop effective therapeutics to patients.

  • Episode Transcript

    Chris Garcia:
    Hello, and welcome to another episode of Vital Science, and thanks for joining us. If you tuned in last month, you'll know we began an exploration of in vitro testing with a focus on CRISPR. If you missed it, I encourage you to check our Vital Science page and download that episode with Drs. David Fischer and Carina Peritore, to hear a fascinating discussion about in vitro gene editing. Just to recap, in vitro is a term that we use to describe the evaluation of biological materials like cells, genes, or other microorganisms in a construct outside of their biological context.

    In drug development, in vitro systems offer a critical platform for understanding the biology of disease, investigating pathways and mechanisms of action, and evaluating the safety and efficacy of new therapies before more costly testing in animal models, or in vivo. And so, especially for contract research organizations like Charles River, there has long been a focus on the development of in vitro technologies that result in the creation of better, more accurate, and relevant assays and models.

    As we've advanced our understanding of human biology, we've been able to develop models that recapitulate both healthy and disease states and build assays that fully interrogate these systems. So, it's a continuous circle of advancement and improvements that are all the time paving the way for more effective therapies and treatments of disease.

    Today, we're going to look at the evolution of cellular models with a specific focus on the development of in vitro 3D systems that are improving our ability to understand and predict how drugs will work within the body. We'll hear a bit about the origins and limitations of dish testing, the driving need for better systems, and the advent of new technology that reconstructs human tissue in a three-dimensional format that gives us more insight into in vivo reality.

    Hailing from a background in big pharma, our guests today are deeply familiar with the discovery research that fills pipelines and brings drugs to market. Dr. Elizabeth Anderson, our scientific director of oncology, and Dr. Ian Waddell, our chief scientific officer sat down with Gina to talk about 3D in vitro models, how researchers are using them, what it means for drug discovery now, and what it might mean in the future. With that, I'll turn it over to Gina.

    Gina Mullane:
    Welcome to the Vital Science Podcast. Today, we're going to talk about a really exciting topic called in vitro 3D models. And there've been many advances in understanding cancer biology, in particular, how cancer cells communicate with each other and other cell types within their microenvironment. This has led to the development of new therapies but has also highlighted the need for more realistic models in which to study biology and test new therapies, especially immuno-oncology drugs. So I'd like to welcome two folks from Charles River to the Vital Signs podcast today. Dr. Elizabeth Anderson is the science director of oncology, and Dr. Ian Waddell is chief scientific officer. Hi, Liz. Welcome.

    Elizabeth Anderson:
    Thank you.

    Gina Mullane:
    Would you mind telling us a little bit about yourself?

    Elizabeth Anderson:
    Yes. I joined Charles River two years ago as science director in oncology. I've always worked in cancer biology, so my first postdoc was in steroid hormones and breast cancer, and I led a translational science lab for many years at the Christie Hospital in Manchester before moving to industry. So I've worked for AstraZeneca and for Boehringer Ingelheim before I joined Charles River. So I have broad experience in research and drug development.

    Gina Mullane:
    Interesting. And so this new perspective of working within a CRO is probably something you're well-equipped to handle.

    Elizabeth Anderson:
    Yes, it's very different indeed.

    Gina Mullane:
    And also welcome to Ian.

    Ian Waddell:
    Thanks, Gina. It's great to be here.

    Gina Mullane:
    So would you mind telling us a little bit about yourself?

    Ian Waddell:
    Certainly. So I have around about 30 years experience in drug discovery, predominantly oncology. I've worked in large pharma, actually alongside Liz at AstraZeneca, but I've also worked in the charity stroke academic setting with cancer research in UK. And I've worked for Charles River for roundabout four years now, initially as the head of biology, but now as the chief scientific officer.

    Gina Mullane:
    Wonderful. Well, thanks for being here. I am a big fan of your social media and encourage others to check out that as well. But let's get into the podcast. So, Liz, this topic of 3D models, could you tell us a little bit about that? What does it mean when people talk about complex three-dimensional models?

    Elizabeth Anderson:
    Hi, Gina. Well, it's almost what it says on the tin. It's a three-dimensional reconstruction of tissues or of cancers that more closely represent the human situation. So development of human cancer cell lines began in the middle of the 20th century when people started exploiting the nature of cancer cells to replicate in an uncontrolled fashion, so they're immortal. And they are developed two-dimensional cultures, by which we mean that these cells were grown on a glass or a plastic surface and it was just a single cell layer. And these were always thought to be pretty cool at the time and they enabled a huge amount of drug development to go on.

    Chris Garcia:
    The creation of the first immortal cancer cell line came from cells taken from the cervical cancer of patient Henrietta Lacks in 1951. Dr. George Gey at John Hopkins discovered that these cells were unlike most, which died within a few days. Gey found that he could reproduce them creating a remarkably durable and prolific resource that the institute began to share freely and widely for research. Having access to an inexhaustible consistent resource of cells allowed scientists to study cancer cell behavior and deepen our understanding of life cycle and function. In the time since, HeLa cells have played a role in numerous scientific breakthroughs including an improved understanding of zero gravity in space, the AIDS virus, leukemia, cancer, and perhaps most notably the development of the polio vaccine.

    Today, there are numerous immortal cell lines including those from naturally occurring cancers and those derived from human stem cells. While two-dimensional cell culture has been around since the turn of the previous century, the discovery of immortal cell lines paved the way for more vigorous research in the '40s and '50s when it became more standard practice. As the bodies of literature grew, more scientists were trained in the technique, which was found to be fairly inexpensive at scale and produced results that were easy to analyze and measure. As a tool for drug discovery, 2D cell culture has been an efficient in vitro system for evaluating drug candidates, but as you'll soon learn, it has its limitations. Dr. Anderson elaborated.

    Elizabeth Anderson:
    These two-dimensional cultures were used everywhere for drug development, for studying biology, but soon became realized that there's a big issue with them. And my colleague, Mina Bissell once said, "It's as plain as the nose on your face, that two-dimensional does not represent the situation in cancers or in normal human tissues, that we don't have all the cell types that are present in a two-dimensional, and we don't have the architecture either." So complex three-dimensional models aim to reproduce the cancer community if we're talking about oncology and also the cancer architecture. So that's what I mean by complex three-dimensional models.

    Ian Waddell:
    Liz, can I just explore that answer a little bit more carefully, please?

    Elizabeth Anderson:
    Yep.

    Ian Waddell:
    Why would people go to the trouble of developing these really quite complex human cell culture systems?

    Elizabeth Anderson:
    Well, it comes down to how representative they are of the human settings. So what we find, particularly in oncology drug development, that a lot of drugs drop out of development in phase two clinical studies. And that's the phase at which you're trying to validate the target, so you've identified a target and you think by inhibiting it or activating it or whatever in a tumor you'll have a therapeutic effect. But actually, we found that a lot of these drugs were failing in terms of efficacy. They were safe, but they didn't do what we thought they were going to do. And that comes down to the fact that we didn't understand how they would interact with cancer cells when cancer cells are in this complex environment, when they're surrounded by other cells, when they've got lots of stroma around them. And by stroma, I mean fibroblasts and protein deposits that encase all these cancer cells. And by not taking all that into consideration, we don't validate the target properly, so we needed better ways of validating targets, and also testing new drugs to see what that complex environment does on drug disposition.

    Gina Mullane:
    So, Liz, as our understanding of how the body works has improved, how has this really allowed us to develop more relevant 3D models?

    Elizabeth Anderson:
    So I guess in the '70s, people started thinking, and I'm going to talk mainly about breast cancer because that's my area of expertise, is that people started to realize that that breast cancer is a complex structure. It's got lots of different cell types, so it's not just the cancer cells, but it also has normal human fibroblasts, it has blood vessel cells, the endothelial cells, and it has a very complex protein surrounding. And people started understanding, so these are colleagues such as Mina Bissell, or the late Zena Werb, and Valerie Weaver began to dissect out how these cancer cells talk to the microenvironment. So how do they talk to the other cells? How do they talk to the stroma? And these advances in understanding the matrices and the cell types have made it possible to start building more relevant, three-dimensional models.

    So moving on from agar, we began to use preparations such as something called matrigel, which is an extracellular matrix that's secreted by a mouse tumor. And this contains all sorts of proteins that we find in the normal tumor microenvironment, so things like fibronectin, collagen, laminin growth factors. And people found that you could grow cells in these matrigel environments and you start seeing organization of tumor cells. And people like Zena Werb and Mina Bissell showed that the extracellular matrix from normal tissue can actually revert the phenotype of tumor cells in some cases. So we started to understand that tumor cells talk to the microenvironment and the microenvironment actually talks back to the tumor cells. So it's what's called, the buzzword at the time was dynamic reciprocity. So there's a two-way talk between tumor cells and their other cells, understanding how cancer cells talk to the protein or the extracellular matrix that surrounded them and also to the other cell type.

    So once we began to understand that, we could start reconstitutes in tumors by putting cancer cells with fibroblasts, with endothelial cells in a complex matrix, such as this preparation called matrigel. And there are other ways of doing this. People have used what's called low attachment plastics, so the cancer cells can't attach to the plastic and flatten out, but they produce these spheroids. And again, we can co-culture with normal cells, fibroblasts with endothelial cells, and also with immune cells in these settings and see how immune cells interact with the tumor cells and how the tumor cells react to the microenvironment. And we can see things like what happens if you'd make changes in the matrix, how does that affect response to immuno-oncology drugs such as antibodies.

    Ian Waddell:
    Liz, can I interrupt and just ask for some more detail in terms of advances that have been made in the types of matrices that have been developed that allow us to look at the microenvironment in more detail?

    Elizabeth Anderson:
    Right. So there's been a lot of advances in matrices, so we have a fuller understanding of what's present in that matrigel that is so good for tumors. So, we know how much collagen is in there, we know how much laminin, and the other components. So one is, understand the matrix, but we can also combine this with three-dimensional patterning techniques, such as the one that's used by our collaborators, Cypre. They have a matrix and they can produce a preparation with tumor cells embedded in a matrix, and then they can add other cells to that matrix, so they can add a fibroblast to that matrix, so you've got fibroblasts and tumor cells. And then on top of that, you can add things like immune cells. So you can take peripheral blood mononuclear cells from human donors and put those into the cultures, and you can ask the question of does the matrix make any difference to how those immune cells will interact with the tumor cells?

    And what we find is that if you have fibroblasts in the matrix with the cancer cells, then the immune cells are excluded. And we've replicated what's called an immune-excluded environment. So we can use these complex mixtures, we can change the cell types within the mixtures, and we can put them in different orientation, so you could have them co-cultured together, you can put cells on the top, and then you can see whether your drug works in that more representative situation.

    Chris Garcia:
    When Dr. Anderson mentioned her colleagues in this space, Drs. Mina Bissell, Valerie Weaver, and the late Zena Werb, I was curious and wanted to know more about them. I did a little research and found that individually and collectively these women's scientists have pioneered our understanding of cancer biology, particularly with the context of the human body. Their concentration on the extracellular matrix that surrounds tumors, what we now call the tumor microenvironment or TME, and the interaction between these cells have paved the way for numerous advances like the creation of more translational in vitro 3D models and the identification of new targets and approaches in oncology drug development. Be sure to check out our show notes if you're interested in reading more about these women and their groundbreaking work. You'll also find more in-depth information on what we now understand about the tumor microenvironment and how to investigate it. Next, we'll find out how the models are created and used.

    Gina Mullane:
    So, Liz, given all of those factors and the complexities, how can we reproduce the 3D structure of normal tissue for pharmacology and toxicology studies?

    Elizabeth Anderson:
    Well, this is really a very exciting area of tissue engineering, as you might want to call it or three-dimensional structures. So in fact, three-dimensional structures of skin cells, where we've got skin cells on a support is old hat in toxicology. So these have been well-validated and well-established for looking at the toxicological effects of various agents on skin cells. But we're now starting to be able to reproduce things like the epithelial membrane or the respiratory epithelial cells. And you can model respiratory epithelium from the upper airway or from the lower airway. And this allows you to look at things like COVID-19 infection, for example, or for other drugs that affect the respiratory epithelial cells, the epithelial lining of the respiratory system. We can do this with other epithelial cells. So people have been looking at oral epithelium and also at reproductive epithelium. So you can start to reproduce some of the normal cells, particularly epithelial cells to look at pharmacology and also for toxicology studies.

    Chris Garcia:
    With the first 3D skin models appearing from skin biopsies in 1948, it's no wonder that Dr. Anderson calls them old hat in toxicology. These have been around a long time and have likewise evolved as technology has grown. The ability to create scaffolds for the 3D cell culture of skin and epithelium she spoke of came from electrospinning in the '80s and '90s, while the more recent biological applications of 3D printing have taken these models to the next level.

    Today, biotechnology companies like Cypre are engineering, unique hydrogel materials like VersaGel to create complex scaffold-free structures for 3D in vitro modeling. You can learn more about this technology in the show notes.

    In this next segment, our guests took the opportunity to discuss 3D modeling in terms of the current drug development landscape, the tremendous potential for these structures to support specific therapeutic areas, and what the future might look like.

    Ian Waddell:
    Sorry, Liz, I was just going to ask for a little bit more detail on an area that's of particular interest to you and I, which is personalized precision medicine, and how will these advances actually help us target those areas?

    Elizabeth Anderson:
    Well, I think the most exciting thing about this is a lot of these three-dimensional cultures or the ability to reproduce tissue structures has been enabled the development or the discovery of induced pluripotent stem cells. So these are stem cells that have been isolated from patients, so from a sample of skin, and of course that represents the genetic background of that patient, so we can take these pluripotent stem cells and then differentiate them to different organs. So you could, for example, differentiate them into nerve cells. And one of the really exciting areas of 3D structure of normal tissues has been the development of brain organoids, where you actually grow complex mixtures of neurons and their supporting cells which have been derived from these iPSCs, so these induced pluripotent stem cells.

    So we can take a patient's sample and we can differentiate into the tissue of interest and you can do this for brain, you can do this for the intestine, you can do it for quite a few different tissue systems, and then you can investigate how drugs might interact that patient's particular genome. So we can take nerve cells from individuals suffering from things like Huntington's disease. If we go back to the cancer model, we can take cancer cells from a patient and test to see which agents that patient's tumor might be sensitive to. We can take normal tissue from that patient and see how the normal tissue might react to the drug regime that you're going to give them.

    So these 3D models offer great opportunities for developing what might be truly called personalized medicine. It's not only what the drug does to the tumor, but it's also what the drug does to the patient's body, so it's highly personalized or can be highly personalized.

    Ian Waddell:
    What about a common feature in oncology, combination screening? Are these models going to be useful in that area?

    Elizabeth Anderson:
    Yes, I think they will because we may want to combine treatments that attack different cell types within the tumor at the same time, or we may want to look at can we sensitize tumors by pre-treating with something, so the ability to grow three-dimensional structures? And we might use tumor organoids in this setting. There are some great advances in developing systems where you can sample at multiple time points, and that will enable us to look at both combination therapists, both when you put the drugs in together, but also when you start to sequence therapies, so you could put one drug in and then the other and see whether that enhances toxicity on cancer cells or not. So what it does, it enables us to determine the effect of the tumor microenvironment on the activity of combinations or sequences of drugs.

    Ian Waddell:
    I guess, Liz, just take that thought a little bit further, for the first time, this allows us to actually target cells outside the tumor in the stroma, for example.

    Elizabeth Anderson:
    Absolutely. Yes, So I think we still have a lot more to understand about what the stroma does, but we do know that this stroma and in particular, fibroblasts in cancers are different from normal fibroblasts. So, if you take fibroblasts from a tumor and grow them, they will be different from fibroblasts taken away from the tumor. So we can start to understand how we can target not only the cancer cells, but the microenvironment that might be supporting their development and also their dissemination around the body, so we know that the matrix has some role in determining how tumors metastasize or spread outside the primary site.

    Gina Mullane:
    Liz, the advances you've been describing are just so exciting. And I'm wondering if you can do me a favor and look to the future. What do you see as some of the exciting things that are on the horizon?

    Elizabeth Anderson:
    So I think the exciting things are going to be automation in terms of being able to grow these three-dimensional models in a reproducible fashion and in high quantities so that we can use them for drug testing. So I think things like the Cypre collaboration that we have will enable that, but there are also lots of other methodologies that are up and coming. So I'll mention very briefly, there's a method you can encapsulate cancer cells and the other cell types in sodium alginate, which allows the structure to develop properly but can be grown in what's called a bioreactor. So that means you can do multiple samplings across time, looking at how those tumor cells change in terms of their metabolism and their response to drugs.

    Moving on, we've got the brain organoids causing a lot of interest at the moment, particularly from the point of views whether there's been a few articles these days about will brain organoids become ventient. And that's almost like sci-fi, that these brain organoids have been shown to transmit synchronized pulses of messages through the neurons, which sort of suggests that they could be consentient at one point, but this is really for the future.

    And on another example is that people have engineered Neanderthal genes into brain organoids and shown that the Neanderthal brain organoids develop differently from those from homo sapiens. So there's some very interesting work going on in the brain organoid area. But there's lots of other advances. There's the organs on a chip where you put small groups, clumps of cells in a microfluidic device, and you can put cells from different tissue types, so they can actually talk to each other on this microfluidic chip. And there's some interest in looking at the interactions between liver cells and endocrine organs in these organs on a chip.

    Other things are intestinal organoids, these are organoids that are developed from the intestinal gut epithelium, and there's interest in looking at the effects of drugs on these intestinal organoids that have been infected with GI viruses. So there's a huge amount of interest in these 3D models, and they have a huge potential in testing new drugs and understanding biology, and in understanding how tissues develop. So going back to the brain organoids, that these small organoids might give some clues as to how the human brain develops in early infancy and throughout life. So there's a lot of exciting things happening.

    Ian Waddell:
    Liz, can I maybe bring us back to what you and I have spent most of our careers working on, which is drug discovery, drug development?

    Elizabeth Anderson:
    Yep.

    Ian Waddell:
    What would you really see as the significance of these complex culture systems in that setting, but also most importantly for the patients at the end?

    Elizabeth Anderson:
    Well, for drug discovery, I think at the moment, I don't think there are 3D complexes are ready for high throughput screening of lots of different drugs. But I think we have a need for them at two specific points. One is when we're trying to validate a target because we need to validate a new target for cancer therapy in the appropriate setting. So if we can do that in a 3D model that closely represents the tumor situation, then reduce the attrition of drugs that go into the clinic and then fail because they have no efficacy.

    But the other end is the patient personalization of medicine. So if we understand how a particular spectrum of mutations, for example, in a cancer cell and how it interacts with the microenvironment, if we understand that, then we may be able to personalize therapies towards patients. And, of course, by looking at the effects of drugs on these more representative models, we may be able to find new markers of both, that will predict response to therapy, but also be able to select patients for therapies. So hopefully, we'll be able to develop more drugs that do not fail in the clinics and more effective drugs, and that we'll understand how to use those drugs in a patient population.

    Ian Waddell:
    So, Liz, that was really fantastic. And you mentioned before, one of the exciting things that you've read recently was about brain organoids. Are there any other really exciting examples that are coming out in the literature at the moment?

    Elizabeth Anderson:
    I think the intestinal organoids, that these have been used quite a lot now in understanding drug development, also in understanding the toxicology on the GI tract. As I mentioned, the respiratory epithelium models are being used for looking at things like COVID-19, how that affects the respiratory system. And in fact, COVID 19 is also being tested in the brain organoids. So there's lots of different things coming through. So there's liver organoids, which can be used for looking at how the liver affects the metabolism of drugs. So there's a lot of interest in several different [inaudible 00:27:02] types. So there's the organs on a chip with the microfluidic devices, which is very exciting. And then there's the what you might call tissue engineering, is that just looking at different ways of producing these spheroids if you like or complex cultures in high quantity and with high reproducibility across an experiment, for example. So you don't want organoids that differ across experiments, but you want them to be reproducible in terms of their response. So lots of things come in forwards and to watch in this space.

    Gina Mullane:
    Liz and Ian, you're clearly experts in this very exciting translational area of in vitro 3D models. We look forward to hearing more about this topic and from both of you in the future. Thanks so much for your time today on Vital Science.

    Elizabeth Anderson:
    It's a pleasure.

    Ian Waddell:
    Thank you.

    Elizabeth Anderson:
    Thank you.

    Chris:
    So we covered a lot of ground today with Dr. Anderson's explanation of the traditional methods of 2D cell culture and its limitations, the evolution of 3D in vitro modeling and its advantages, new techniques, and what these applications mean for current and future drug development. As you heard, Dr. Anderson and Dr. Waddell both added a lot of value to the conversation.

    We'd also like to recognize our partner, Cypre, the creators of the VersaGel patterning technology and makers of the Falcon X in vitro 3D modeling platform for the evaluation of cancer therapeutics. It was interesting to hear how valuable such systems can be for screening drug candidates in an accurate disease-relevant model of the tumor microenvironment. If you want more information on how it works specifically, you could find this in our show notes.

    We hope you enjoy today's discussion. And for those looking to delve deeper into the topics of today, please review the show notes on our episode page. If you have any questions, please send them along to [email protected] Until next time, thanks from all of us at Vital Science.