Industry Insights with Crown Bioscience: In Vitro Technologies for Studying the Cancer-Immunity Cycle

This episode of Share Science features Rajendra (Jen) Kumari, PhD, the Executive Director of Integrated Solutions at Crown Bioscience, and Gera Goverse, PhD, the Director of Immuno-Oncology. Recently, Jen and Gera presented a webinar that covered in vivo, in vitro, and ex vivo technologies and methods that can be used for immuno-oncology research along each step of the cancer-immunity cycle. In this interview, they take a deeper dive into these concepts and address some of the best questions that were asked by the audience.

What is the cancer-immunity cycle?

Jen: The cancer-immunity cycle basically describes the necessary steps needed for our immune system to effectively eradicate cancer. There are a lot of genetic alterations within cancer which generate tumor-specific antigens that are very distinct from normal tissue. This is sufficient to trigger a cascade of biological events, starting with the presentation of those novel unique antigens on cancer cells to T cells. Those T cells are then activated so that when they reach the tumor microenvironment (TME), they can recognize tumor cells and effectively kill them. More antigens are then released and the whole cycle starts again.

“[The cancer-immunity cycle] is a very highly regulated process, and it prevents the recognition of self- and autoimmunity.”

There are numerous checkpoints. You’ve got PD-1, PD-L1, and CTLA-4. You’ve got a range of different stimulators, so CD40, OX40, and CD137, and then many different types of inhibitors such as cytokines. These work to keep that balance, but this also means that there are many points within that cycle that can be impaired or dysregulated, resulting in tumors escaping immune surveillance and disease progression.

“This cycle captures very nicely, I think, the potential therapeutic targets that can be modulated to get that cycle back on track, as well as help us determine why there may be some therapeutics that are not working.”

I think, in summary, that the cancer-immunity cycle really describes the biology very well, but also how it can be very ineffective in cancer, allowing that disease progression. It also informs how immune therapeutics can be effective or ineffective.

How can you assess the steps of the cancer-immunity cycle?

Jen: There are many different assays and model systems, and it’s really hard to describe everything. Generally, you can model that cancer-immunity cycle or parts of it in vitro and in vivo, which depends on the biology of the target and the therapeutic being assessed. You have to kind of design your assays and investigations based on those key points.

When we are using in vivo systems, for example, then ideally a fully functional immune system would allow the whole cancer-immunity cycle to be assessed, and the endpoint would be tumor killing to demonstrate efficacy. These systems can include genetically engineered mouse models where tumors arise as a result of engineered oncogenic drivers, or syngeneic or homograft models. The tumor develops over time in these models, and so they can then grow and avoid immune surveillance.

“Any successful therapeutic intervention will result in that tumor reduction.”

Then, we can take samples at different stages from that study and look at tumor infiltration or cytokine release to see whether that response has worked or why it might not have worked. We do have to address the limitations of these systems. I think the biggest limitation is that we’re looking at murine-based biology with these models, so any hypothesis that we may generate from studying and using these models needs to then be translated into the human system.

“We still need to have further investigation into humanized systems.”

Again, this is possible with in vivo models, but is very complicated and time-consuming. We have to have alternatives. I think this is where in vitro systems really come into their own. We can do in vitro human systems as well, so you can overcome some of those limitations and really break down the cancer-immunity cycle so that you can look at different stages as well.

Gera: I totally agree with you, Jen. Indeed, you can really analyze more specific parts of the cancer-immunity cycle with in vitro assays instead of just looking at the overall complexity of the whole system in vivo. In vitro systems allow you to work with the human system, and you can incorporate the immune compartment in perhaps a better setup than within some of the mice models.

Additionally, in vitro models allow for higher throughput screening. If you would like to test combinational effects or different doses of your therapeutic compounds to optimize their efficacy, that’s really something that can be addressed with in vitro systems. For example, it can be done in 2D cell cultures, but those are just really high throughput solutions and can give you some simple answers, I would say. If you go to a more complex system with 3D cell cultures or incorporating organoids or patient tissue, that would really add a higher value for clinical translation.

“Assays can really bring [you] a step closer to clinical trials.”

Jen: These systems really work in concert, don’t they? They really do help support, so you can go back and forth as well and dive deeper into the different questions. Certainly, I think combinational effects is a very good example because you just can’t simulate all the different combinations in vivo as effectively as you can in vitro.

In vitro models really help reduce the number of animals we’re using in the end, and then also help us decide which ones to take forward and which to investigate further. Some of the technological advances as well, such as 3D modeling, have taken it a step closer to being more relevant.

“As technology and techniques improve and get better, we get better at modeling cancer.”

How are organoids are impacting the field?

Gera: During our webinar, we polled the audience and noticed that almost 50% were not yet familiar with organoids. I think it was very good that we had that crowd on the call because I think organoids could really be the future for 3D cell cultures.

Organoids are patient-derived, and unlike cell lines, they’re not immortalized. By providing the right growth factors, you’re stimulating stem cell growth and capturing tissue heterogeneity so that those stem cells can grow into different subsets of cells. You are not just looking at one homogeneous population like with cell lines, which can give you valuable input on specific cellular responses.

“I think the heterogeneity in organoids is really bringing the value of getting a better understanding of how patients will [react].”

In the webinar, we showed that many of the techniques out there are making use of imaging-based technology, which brings added value because you can really observe morphological differences. In addition to looking at tumor tissue, you can image immune cells and analyze their positions relative to tumor tissue. This is something that can only be captured through high content imaging (HCI). I think that our HCI platform complements the use of organoids. With high throughput imaging platforms, we can analyze treatments and combination therapies in one big go.

“Coming back to our previous point … there will always be a combination of in vitro and in vivo assays.”

What’s maybe interesting to mention is that we can also have patient-derived xenograft (PDX) organoids. Patient tissue has been used for in vivo models, but from those, we also have the organoids. We can first screen using organoid models, and then follow up with in vivo models.

What is the difference between using organoids versus ex vivo patient tissues?

Gera: As I mentioned, organoids are patient-derived and consist of a heterogeneous population of cells, thereby providing higher value for preclinical testing. In contrast, the ex vivo patient tissue platform is really fresh patient material (e.g., biopsies, ascites, pleural effusions) from clinics. I think it’s really important to note that this material is not pre-culture. From that material you can also make organoids, which could be interesting.

The patient material contains this native TME, which is really the difference. With organoids, you can really stimulate stem cell growth, but in doing so, you lose other cellular players in those cultures such as stromal cells or immune cells. This native TME is maintained in those patient tissues, and I think the complexity of the tissue is maybe much higher. It’s almost as if you are doing in vitro clinical trials in a Petri dish on a high throughput platform. You can actually test multiple treatments or combination treatments within one “patient.”

“We call it an in vitro clinical trial, which is really valuable, again, for later translation to the clinics.”

Do you think there’s been a shift to using more 3D cultures?

Gera: If you look at the poll again, approximately 50% of the audience indicated that they are still working with 2D cell cultures. Maybe I’m a little bit biased, working in a 3D in vitro type of platform at Crown Bioscience. On the other hand, it’s interesting to see that the other half of the audience is already working with 3D cell cultures, which on their own have been on the market for quite some time. I think organoids were introduced about a decade ago, and there has been an increase in the number of publications that demonstrate their clinical relevance.

With organoids, you can perform or analyze more treatments again, combinational treatments. You are working in a relevant system. You can make it as complex as you want by incorporating different immune cells and other cellular players, so you can really reconstitute the TME within this screening-based platform.

“I really think that this will be where everyone in the end will be moving forward.”

Jen: Those are very good points. Certainly, I’ve seen shifts away from 2D cell culture. There’s a lot of criticism surrounding cells that are grown in 2D. Even when you move them into xenograft systems, they form cancers which don’t represent the original cancer type. I think 3D cultures take those cells and put them into more physiologically relevant types of environments, which means that they remember their origins.

“[3D cell cultures] do represent the tumor in the end far more faithfully than the 2D cells.”

We’ve seen collections like the NCI-60 panel being retired in favor of more clinically relevant systems. Certainly, that is where patient-derived systems are coming through. I think the challenges that we’ve had with patient tissue (e.g., access to tissue, use of tissue) can be overcome with these systems, and that really makes them more efficient, cost-effective, and translationally valuable. I think there will be more adoption. We’ve had more and more people asking us for these types of systems.

Gera: Although there aren’t many publications on it, we do know that there are indeed compounds that went to clinical trials that were showing really good results in preclinical testing but failed later on. There have definitely been some failures in clinical trials due to a lack of a certain expression or sensitivity by not using the right preclinical model.

Why should drug developers work with Crown Bioscience?

Jen: We offer, as you’ve heard, a broad range of capabilities for oncology and immuno-oncology including biomarker analysis and bioinformatics. We have a longstanding track record in working with drug developers across pharma and biotech for these preclinical studies. We’ve investigated many different types of therapeutics (e.g., immune checkpoint inhibitors, cellular therapies, oncolytic viruses, vaccines, gene therapies, microbial modulators), as well as resistant mechanisms and those combination strategies that we talked about.

We also demonstrate this experience and capability by sharing the data that we’ve generated, as well as how we’ve characterized our models, which you can access through our website and databases. There are a lot of molecular features. For patient-derived models, we’ve got clinical annotation and relevant data.

“I think, as highlighted already, those patient-derived systems for in vitro, in vivo, and ex vivo are really a unique capability.”

The ability to then run those clinical trial-like studies in those different platforms is making a big impact, I think, and is certainly an area that we’re very committed to developing further. I think there are a lot of capabilities on which we can provide a lot of expertise, and we can guide collaborators on which platforms and systems are best for a particular therapeutic.

Gera: Maybe just to add onto that, I think through the synergy of the in vitro and in vivo models, we can actually nicely follow up with those models. We have experts in both fields, and so we can really start from the beginning. We gave an example in the webinar where we were involved at the beginning stages of identifying hit compounds all the way through to the end, which led to our collaborators sending a compound into clinical trials. I think that shows that we are not just one part of the cancer-immunity cycle that you can address: we’re really the full package, from the beginning until the end. I think that’s really a value.

If somebody wanted to get in touch with Crown Bioscience, what should they do?

Jen: You can start with our website, where we have a whole range of resources. If you need more information, there are factsheets, white papers, publications, and presentations. You can really get a lot more detailed information just by accessing those resources. Then, if you need to ask any questions, you can just contact us via the website as well. You can also register for different databases like for organoids, PDX models, murine systems, or our cell line models. We highly recommend visiting that website and reaching out to us as soon as you have a question.

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