The Interrogation Room
Collecting evidence on the immunological makeup of syngeneic tumor cell lines could be uniquely valuable in cancer immunotherapy.
There's no question that cancer immunotherapy is the current "Big Thing" in oncology research. (Check out Eureka's What's Hot in 2015). The number of approved and experimental drugs designed to enlist the help of the immune system in fighting both blood and solid tumors continues to grow, and some analysts predict that by 2023 up to half of all cancer treatments will be immunomodulatory.
But it's still early days. We still have a lot to learn about the immune responses induced by these pharmacological agents and, more importantly, what the long-lasting effects are on both the patient and the tumor. To paraphrase Albert Einstein, "The more we learn, the more we realize how much we don't know."
To enhance our understanding of this incredibly complicated and heterogeneous disease, researchers have seized on an easily accessible research mouse model that offers something other models cannot—an intact immune system. Allograft mouse tumor systems, otherwise known as syngeneic or syngeneic models, are tumor models whose genetic background is similar if not identical to the host animal.
Because they retain intact immune systems, syngeneic mouse models representing a variety of hematological and solid tumors—leukemia, melanoma, lung, colon and breast cancer to name a few—can be particularly relevant for studies of immunologically-based targeted therapies, either used alone or in combination with other drugs that modulate the immune system's ability to seek out and destroy cancer cells (see Oct. 30, 2013 Eureka blog).
Scientists began developing syngeneic models over 50 years ago, mostly from carcinogenesis studies or exploitation of spontaneous cancer-causing mutations. For example, the Lewis Lung cell line was isolated from a spontaneous lung carcinoma from the inbred C57Bl/6 mouse. It was used extensively in early chemotherapy research and is presently employed through subcutaneous, intravenous or intraperitoneal inoculation. Likewise, Colon26 was isolated from the colon of a BALB/c mouse in a carcinogenesis study, and was also commonly used in the early days of cancer research.
A key limitation of syngeneic models is the lack of human targets. With the evolution of structurally targeted therapies, syngeneic models moved to the periphery of oncology drug discovery in favor of human xenograft models and genetically engineered mouse models which present the human target. However, syngeneic models have recently resumed center stage after having been identified as reliable models for immunotherapy studies. There is now a great deal of interest and effort at Charles River Discovery Services and elsewhere, to "profile" as many of these syngeneic cell lines as possible in hopes of steering research questions to the right model.
So what do we mean by profiling and what does it involve? Well, it's actually a way of interrogating a model to obtain information. It is very similar to a forensic scientist who conducts DNA profiling to collect evidence for criminal trial. However, through these profile studies, we collect evidence on the immunological makeup of these tumors and how they respond to targeted immunotherapy. Many of the syngeneic models are well-characterized, validated and available for study, but once the field began exploiting these models for immune-based therapy, it became clear that there is still a lot to learn.
Our profiling efforts began over a year ago, when we identified over 30 syngeneic tumor cell lines based on popularity of these models in the published literature. Several of these lines were already in use in our labs, and eight more cell lines were added to our portfolio. Histotypes currently include breast, lung, colon, leukemia, lymphoma, mastocytoma, plasmacytoma, pancreatic, melanoma and renal, and we hope to add at least five more by the end of this year.
We then scanned the literature to find the most appropriate host strain for these new lines. In the majority of cases, we use the same host strain that the model was derived from—the EMT-6 breast tumor cell line from the BALB/cCrgl mouse being one example. We do this to minimize the chance of host rejection and to provide the most advantageous environment for tumor growth.
Next, we inoculated two to three cell concentrations into the host to get a sense of the percentage of tumors that form following inoculation as well as growth rate of those tumors. Some tumors grow fast and others slow. Even syngeneic tumors can be variable, so it is important to understand individual growth kinetics because it can impact the study design.
We are currently looking at how a handful of these models perform against a trio of monoclonal antibodies against immune checkpoints, a class of protein targets that act as a kind of brake by shutting down T cell immune responses and preventing the immune system from going rogue. Two colon tumor models, Colon26 and MC38, are particularly strong responders to checkpoint inhibitors. The EMT-6 breast tumor model also responded relatively well. Other lines, including B16F10 melanoma and Lewis Lung did not respond well, suggesting they would be suboptimal choices to test this type of targeted therapy without additional manipulation.
So what have we learned? We have learned that the timing of treatment is important. In some cases, if a syngeneic tumor-bearing animal is dosed a few days after implant, the response can very strong. However, if the same treatment is delivered just a couple of days later, the tumor progresses as if the animal was not dosed at all.
We also found that specific clones of these antibodies directed against these checkpoint inhibitors, which are directed to different epitopes on the same target protein, can alter the strength of the anti-tumor response. These responses can vary widely within the same tumor model from very little response to a virtual cure. You can imagine that combination strategies are influenced by this finding. We have further investigated these differential responses by treating even larger tumors with the most active antibodies and by reducing the antibody dose to turn that strong response into a moderate response. These regimens could then be combined with our client's agents in hopes of a synergistic response.
We're now also looking closely at the tumor-infiltrating lymphocytes from these models to gauge the presence of immune cell populations such as regulatory T cells (Tregs), effector T cells, and myeloid-derived suppressor cells (MDSCs). Once we have a better idea of what immune cell populations are localizing to these tumors, we can then add the checkpoint inhibitors and other agents and monitor how these populations are altered with treatment. This new capability will be a powerful tool to help directly understand how the immune system modulation impacts tumor growth.
Cancer is a tough disease, so the weapons need to be tougher. By developing immunological profiles of syngeneic tumor models, we can provide the necessary base of knowledge for drug developers to build a new arsenal of cancer therapies.