The human immune system is highly complex. Not only is it made up of millions of different cells, but it also varies from person to person. With progressing age, environmental disturbances or the onset of disease, the immune system’s responses change.
“In cancer, for example, the immune system is disabled within the tumor microenvironment,” says Dr. Rajendra Kumari, Global Head of Scientific Communication at Crown Bioscience. “In this instance, the immune profile is altered such that it will help the tumor as well as protect it. The tumor becomes invisible to the immune system, which is why some cancers progress so rapidly.”
The ability of some tumors to remain undetected by the immune system is also the reason why some immunotherapies, such as checkpoint inhibitors have experienced a rise in popularity within the clinical oncology landscape.
Checkpoint inhibitors such as Keytruda® (pembrolizumab), which targets PD-1 and Yervoy® (ipilimumab), which targets CTLA-4, have the ability to switch the immune system back on and can make the tumor detectable again. Whereas they and other immunotherapies have revealed significant efficacy in some patients, many others experience remission, relapse or resistance to cancer treatments.
“We are trying to understand why this happens to ensure that we see further clinical success with these agents and to look for ways to broaden the patient populations that will benefit from these immunotherapeutics,” Kumari explains. “For this purpose, we use immune profiling as a way to measure the state of an individual’s immune system at any given point in time.”
Understanding tumors and immune therapies with immune profiling
In the past, Kumari says, researchers focused mainly on the tumor and the study of different malignant cell types prior to and following treatment. Now, however, it is also important to analyze what happens with the immune cells within the tumor microenvironment.
“Immune profiling has really changed the way we look at cancer and how we treat it,” says Kumari. “There’s a lot of benefits to immune profiling as well. One of them is the discovery of new targets within the tumor microenvironment, allowing for the development of new therapies and different approaches to tackle cancer.”
Immune profiling allows researchers to study the composition of the immune system, it can help them to define which tumors may respond to a specific treatment and which may not. For instance, some tumors are characterized by a high T cell infiltration and are more likely to respond to certain therapies, whereas others have scarce T cell infiltration and are less likely to respond. With the help of immune profiling, these tumors can be identified and characterized.
“There is a strong chance of survival in patients with tumors that have high counts of CD8+, Th1 or PD-L1. On the other hand, high presence of suppressive myeloid cells is correlated with a poor prognosis,” Kumari explains. “So immune profiling can help us identify biomarkers to predict which patients will respond to treatment. This can save a lot of valuable time in the clinic, especially for those patients who won’t benefit from these specific immunotherapeutics, who may benefit from alternative therapies.”
Syngeneic mouse models to observe tumor-immune system interactions
One of the main challenges in immune profiling, says Kumari, is the fact that there is still a lot to learn about the microenvironment of tumors and the interplay between different cell types, which can sometimes make it hard to decide which treatments to use. Here, syngeneic mouse models can be of help.
Syngeneic cell lines are derived from a range of different tumor types, such as prostate or colon cancers. These cell lines are then implanted into mouse hosts with the same genetic background and a functional immune system.
“Once the cell lines are implanted, it becomes apparent that each model modulates the immune system differently. In other words, when we examine the immune profile of one particular cell line, we can see that it’s different from that of other cell lines,” Kumari says. “So we can see that the immune profile is very much driven by the tumor type and we can see a wide diversity within syngeneic models.”
There are a lot of advantages in using syngeneic models. First, they are easy to set up and very cost effective. Moreover, they are typically used for preclinical proof-of-concept studies and allow researchers to study the efficacy, mechanism of action, and the pharmacodynamics of an agent. “Syngeneic models can also be benchmarked against those checkpoint inhibitors already used in the clinic, so researchers can see how well their agent is working in comparison to those benchmark agents,” Kumari adds. “And they can be used to observe the effects of combinational therapies.”
Syngeneic models can help researchers to translate results into the clinic
Syngeneic mouse models allow researchers to study the interaction between different tumors and the immune system, as well as the effects of immunotherapies on the tumor and surrounding immune cells. The models reflect the diversity of immune profiles that are also found in the clinic.
“When we do immune profiling, we can see that the range of immune cell types that infiltrate different tumors varies. For example, popular models, such as MC38 and CT26 have a very high amount of infiltrating CD8+ T cells. This suggests that the immune system is just switched off, therefore a checkpoint inhibitor can release the brakes and activate these cytotoxic T cells within the tumor microenvironment to kill the tumor or these models can help understand why the tumor may not be killed,” says Kumari. “This can translate into the clinic as seen by the development of checkpoint inhibitors such as Keytruda®.”
Other models, including the melanoma line B16-F10, have very low levels of infiltrating T cells. Even if a checkpoint inhibitor were to activate the T cells within the tumor microenvironment, there would not be enough to mount an attack on tumor cells. This also reflects the high diversity within patient populations – differences from patient to patient, tumor to tumor, and even among patients with the same tumors.
How CrownBio tackles patient population diversity
“At CrownBio we understand that there is a need to replicate the diversity within the patient population, so we invest quite heavily in building large collections of cell lines and syngeneic models,” Kumari explains.
“In our portfolio, we have 48 different syngeneic lines. We provide a large amount of immune profiling data for our in vivo models, as well as RNA sequencing data, growth profiles, response to chemotherapeutics or checkpoint inhibitors. All of this can be viewed via our online database, which can help researchers to choose the right model for different targets or immunotherapeutics. And we have set up operational sites all over the world, allowing clients to bypass the challenging shipment of novel immunotherapeutics and to run their tests locally. We also have a large scale in vivo screening platform enabling large numbers of models to be tested at once.”
Furthermore, the company has picked up on the fact that syngeneic mouse models – though helpful to study tumor and immune system interactions – are not completely reflective of human disease. They are based on spontaneous tumors that have arisen in mice and are grown in tissue cultures that have inevitably adapted to plastic. This removes traditional syngeneic models even further from clinical relevance.
Introducing the murine tumor homograft model
“If you look at pancreatic cancer, for instance, the majority of pancreatic cancers are driven by K-ras mutations,” Kumari explains. “But the available syngeneic pancreatic cancer lines actually don’t carry that mutation. It is therefore not a very representative model.”
To circumvent this issue, CrownBio partnered with the Shanghai Model Organism Center to develop a mouse model called the murine tumor homograft model, which uses tumors from genetically engineered mouse models, referred to as the ‘patient’, that are transplanted into the murine host. “This method allows us to do efficacy testing more streamlined, and these tumor cells are in the mice and have never been in contact with plastics, so they retain most of their molecular phenotype and architecture,” Kumari says.
In the example of pancreatic cancer and K-ras mutations, these murine homograft models actually carry the specific mutation and develop the disease in the pancreas. The researchers at CrownBio then move these tumors into a subcutaneous environment so they can be used for screening. “This way, we’re not only looking at the specific immune environment of a tumor but also at a tumor with a very specific mutation,” Kumari concludes.
Using bioluminescent imaging to observe tumors
Although the subcutaneous site allows researchers to monitor the tumor implant and its growth, it does not fully mimic the organ-specific environment with the relevant vasculature, the organ typical interactions, environmental pressures, and oxygenation. However, capturing these different parameters is essential because they influence how the immune cells infiltrate into the tumor microenvironment, which affects tumor growth within the organ and drug sensitivity.
So in order to observe the tumor inside the organs, the researchers at CrownBio use bioluminescent imaging to track the growth and development of the tumors inside the relevant organs.
“We apply bioluminescence for all orthotopic models because it has a high throughput and is user-friendly. We do this by labeling the cell lines with a bioluminescence reporter called luciferase, which makes the tumor visible by emitting light,” Kumari explains. “That way we can track the tumor in the organ, look at metastases and quantify lesions. It also shows us what size the tumor is and whether it’s ready to recruit to the study and start with the treatment.”
Combinations are the way forward
Syngeneic models can be widely used to evaluate many different types of immunotherapeutics such immune modulators, bispecific antibodies, tetramers, vaccines, viruses, bacteria, cellular therapies and more. These agents can be evaluated as monotherapies but also as combination regimens with other immunotherapeutics, as well as in combination with chemotherapy, radiotherapy or other targeted agents.
“Most success will be seen with combinatorial approaches,” Kumari says. “There are numerous clinical trials ongoing looking at combinations with Keytruda® for example to enhance the T cell activity, promote T cell trafficking into the tumor or increase the immunogenicity of the tumor. So having a preclinical platform to profile these combinations before they enter the clinic is very critical, and syngeneics or homografts are the go-to models for preclinical research.”
Images via Meletios Verras, Sebastian Kaulitzki, Andrea Danti, Kateryna Kon, Lightspring/Shutterstock & Crown Bioscience
Author: Larissa Warneck, Science Journalist at Labiotech.eu