Guest Commentary: Preclinical studies gain the edge with syngeneic models
April 2018
by Dr. Megan MacBride of Taconic  |  Email the author
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Sometimes, the solution to a challenge is not the newest advancement but a tried-and-true approach that’s proven effective for decades. Such is the case with the use of syngeneic tumor models in certain preclinical research applications. Used for more than 70 years, syngeneic mouse models are employed extensively in early-stage drug discovery and have become a workhorse for one of the fastest-growing areas of research: immuno-oncology. These well-documented models are transforming how investigators approach novel therapies, especially immunotherapies.
 
The right foundation
 
A syngeneic tumor model is developed by deriving a tumor cell line from a mouse model of an inbred strain, growing the tumor cell line in vitro and implanting the tumor cells into a mouse of the same inbred strain. The requisite cell lines are available for a wide variety of tumor types and can be cultured and expanded easily, while the inbred strains required as hosts are relatively inexpensive and readily available in study-appropriate quantities. For these reasons, syngeneic models are often used in early-stage drug discovery as a screening tool.
 
The use of syngeneic tumor models yields a number of advantages for researchers, particularly those studying immuno-oncology. The fact that the tumor cells and host mouse are genetically identical improves research into interactions among tumor cells, immune cells, the associated stroma and humoral effects, as they are all derived from the same species, in contrast to xenograft tumor models in which the mouse host stroma and remaining immune system interact with human tumor cells. Since the hosts used to develop syngeneic tumor models can be fully immunocompetent—possessing an intact and functional immune system, as well as the cells and tissues necessary to initiate a tumor-immune response—these models facilitate the study of tumor-immune system interactions and immunotherapy efficacy.
 
In the 1970s, syngeneic tumor models fueled important early discovery work that elucidated the role of T cells in cancer development and treatment.1 Today, these models are again transforming oncology research, with groundbreaking studies on checkpoint inhibitors, research on immuno-oncology combination therapies and investigations into the role of the microbiome on immunotherapy—all employing syngeneic tumor models.
 
Breaking ground
 
Two of the earliest studies that demonstrated the powerful potential of checkpoint inhibitors used syngeneic tumor models to validate these novel immuno-oncology targets. A study by Jim Allison’s lab used the 51BLim10 cell line in BALB/c mice to demonstrate that inhibition of CTLA-4 was a viable strategy to enhance antitumor immune responses.2 Such studies eventually led to Bristol Myers Squibb developing its anti-CTLA-4 drug Yervoy (ipilimumab). Merck used the syngeneic MC38 colon cancer line in C57BL/6 mice to validate the strategy of antibody-mediated inhibition of PD-1, which was successfully brought to the clinic as the blockbuster drug Keytruda (pembrolizumab).3 These immune checkpoint inhibitors are now widely used as efficacious immunotherapies.
 
More recently, syngeneic tumor models have proven vital in preclinical research into oncology combination therapies. Such combination therapies may extend the utility of immunotherapies to a broader patient population.
 
A series of Massachusetts Institute of Technology studies explored the effectiveness of a combination therapy in a syngeneic tumor model of melanoma. The use of syngeneic models was critical to this research, as the successful action of the combination therapy relied on response from multiple immune cell types, including both adaptive and innate immune cells. The majority of mice engrafted with syngeneic melanoma tumors were cured of the initial tumors and protected against future attempts to reintroduce the same tumors, suggesting this combination therapy approach could prevent cancer recurrence.4
 
The gut’s role
 
Syngeneic tumor models also are helping investigators explore how the microbiome affects both tumor growth and the host’s response to immunotherapy. Since the inbred mouse strains suitable for use as syngeneic tumor models are available at a wide range of health standards, including germ-free, they are well suited to preclinical studies involving the microbiome.
 
CTLA-4 blockade therapy is one approach in which the treatment has been shown to have significant positive results in some patients, while in others it has proven ineffective. A 2015 study in Science sought to explore the role of the patient’s microbiome on this immunotherapy. The authors found that although CTLA-4 blockade controlled tumor growth in mice with a conventional microbiota, it was not effective in germ-free mice. Specifically, the antitumor efficacy of CTLA-4 inhibition was found to be dependent on certain species of Bacteroides, which had an immunostimulatory effect.5
 
Another study in the same issue of Science investigated the role of different microbiota on melanoma growth, using B16.SIY tumor cells in C57BL/6 mice. The researchers discovered that mice from different vendor sources, harboring different intestinal microbiota, displayed varying degrees of spontaneous antitumor activity.6 These differences were eliminated after fecal microbiota transplantation was performed on the mice or after they were cohoused to facilitate microbiota transfer. The authors identified Bifidobacterium as the agent responsible for the difference in tumor control. Administration of Bifidobacterium was as effective in improving tumor control as use of a PDL-1 checkpoint inhibitor, and combination therapy consisting of Bifidobacterium administration and anti-PDL-1 antibody treatment gave improved tumor control over either monotherapy.
 
These studies and others indicate that manipulation of the microbiome may be a valid therapeutic approach to enhance the efficacy of immunotherapies and/or achieve response in otherwise non-responding patients.
 
With the microbiome’s impact on tumor growth and antitumor activity so well documented, it is critical to consider the health standard of the model chosen to serve as host when using syngeneic models in oncology research. Most inbred mouse strains are available at an SPF (specific pathogen free) or SOPF (specific and opportunistic pathogen free) health standard, though the particular excluded pathogens and opportunistic pathogens will likely vary by vendor. A limited number of strains are available at the germ-free health standard. Maintaining consistent health standards across studies is essential to improving study reliability, making it vital to adhere to strict barrier conditions and follow consistent SOPs. Ordering mice from the same production location and using the same animal room for experiments over time also are best practices.
 
In addition to selecting the most appropriate health standard for a syngeneic tumor model host and maintaining that health status over time, it is equally important to select the most appropriate tumor cell line for a study. More than 100 tumor cell lines have been developed since these models first came into use. To date, researching the available options has been a manual, time-consuming endeavor. To simplify the process for investigators, Taconic Biosciences recently completed development of a unique database of tumor cell lines for syngeneic studies, compiling 110 such cell lines. This reference tool can be filtered or sorted by tumor type, originating inbred strain, immunogenicity, metastatic potential or cell line, greatly streamlining the process of finding and selecting the most appropriate tumor cell line for a syngeneic tumor study.
 
Syngeneic tumor models will continue to aid preclinical research, providing a well-documented platform on which to conduct target screening and other early-stage drug discovery. And given their unique ability to facilitate the study of tumor-immune system interactions and immunotherapy efficacy, syngeneic tumor models will continue to serve as a workhorse of immuno-oncology research.

Megan MacBride, Ph.D., is director of commercial models for Taconic Biosciences.
 
References
1. Berenson, JR., et al. Syngeneic adoptive immunotherapy and chemoimmunotherapy of a Friend leukemia: requirement for T cells, J Immunol. 115 (1): 234-8 (1975).
2.Leach, D. et al. Enhancement of Antitumor Immunity by CTLA-4 Blockade, Science 271 (5256): 1734-6 (1996).
3. Food and Drug Administration Center for Drug Evaluation and Research (2014) Application Number: 125514Orig1s000. Cross Discipline Team Leader Review.
4. Moynihan K., et al. Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses, Nat. Med. 22 (12): 1402-1410 (2016).
5. Vetizou, M. et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota, Science 350 (6264): 1079-84 (2015).
6. Sivan A., et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy, Science 350 (6264): 1084-9 (2015).

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