Profiling TILs in Syngeneic Mice to Support Drug Development

Jul 8, 2021 11:00:00 AM / by Champions Oncology

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Mouse models have been essential to the development of novel immunotherapies that target solid tumors and hematological malignancies. Several types of mouse models exist, and syngeneic mouse models offer several advantages for screening potential drugs and biologics since tumors are derived from the same genetic background as the mouse strain. Syngeneic tumor models are particularly useful for profiling tumor infiltrating lymphocytes (TILs) that are found within the tumor microenvironment (TME) of solid tumors.

Highlighted below is how immune profiling of TILs can be carried out in syngeneic mouse models and how this informs preclinical studies.

What are syngeneic mouse models and how are they made?

Syngeneic mice are immunocompetent mouse strains that can be transplanted subcutaneously with an MHC (major histocompatibility complex)-matched tumor cell line, such as the widely used melanoma cell line B16F10 or the colon carcinoma cell line CT26. These transplanted tumors grow larger over several weeks, and mice can be treated systemically or intratumorally with experimental drugs or biologics. Syngeneic models offer easy access to tumors, but they are not located in the appropriate anatomical location with respect to the tumor of origin, thus limiting the interpretation of some tumor-specific responses to experimental treatments. The rapid growth kinetics of tumors in syngeneic models can make it difficult to evaluate biologics that require a longer treatment window, and this type of model is not suitable for measuring the efficacy of a treatment during early tumor development[1]. But syngeneic tumor models have intact immune systems, which is not the case for patient-derived xenograft models that have suppressed immune systems that can tolerate human tumor grafts. The mouse lines and tumor cell lines used for syngeneic models are widely available and may be a more affordable and accessible approach than xenograft models. In addition, the precise delivery of tumor cell lines to syngeneic mice can yield more consistent tumor growth compared with genetically modified mouse lines that carry oncogene or tumor suppressor mutations or carcinogen-induced tumor models.

How do I profile immune cells within tumors?

Subcutaneous tumors can be excised relatively easily in syngeneic mice, and immune profiling of TILs can be done using multiple methods that include traditional flow cytometry, single cell transcriptome analysis, mass cytometry/CyTOF (cytometry by time of flight), and immunohistochemistry (IHC)[2]. Flow cytometry and immunohistochemistry are well established methods that have evolved over time. Flow cytometry analysis of single cell suspensions derived from tumor tissue requires staining with fluorescently labeled antibody panels followed by sample analysis on a flow cytometer. Reagent and hardware advances allow for the detection of multiple lymphocyte subsets and polymorphonuclear cell subsets, including tumor infiltrating lymphocytes and monocyte derived suppressor cells (MDSCs). Mass cytometry or CyTOF is a method with similarities to flow cytometry and is used to quantify proteins in individual cells2021Jul08_BlogInside through a process involving labeling with antibodies conjugated to heavy metals. CyTOF data can be used to characterize the TME by detecting and measuring different cell subsets as well as changes in activation marker expression[3]. Advances in automated tissue processing and high-throughput image analysis methods has brought immunohistochemistry into the 21st century. IHC is now used to track changes in biomarker expression in tumors from different preclinical or clinical treatment protocols[4]. More recently, single cell RNA sequencing methods have become widely used for profiling changes in the TME related to treatment, and analysis of this transcriptomic data in combination with flow cytometry, IHC and CyTOF is providing insights into characteristics in the TME that promote or limit anti-tumor responses[5]. Loss of cytotoxic anti-tumor TILs, enrichment of suppressive cell subsets, like regulatory T cells and MDSCs, or the absence of immune checkpoint molecule expression like PD-1/PD-L1, are primary causes of treatment failure that can be identified using these methods in syngeneic mouse models and are being targeted for improved immunotherapy strategies to render tumors susceptible to anti-tumor responses.

Syngeneic mouse tumor models are a useful tool for preclinical oncology research and are especially useful for immune profiling of the TME under different treatment conditions. Consider incorporating these models and immune profiling methods into future preclinical studies of potential drugs or biologics.

 

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[1] Chulpanova DS, Kitaeva KV, Rutland CS, Rizvanov AA, Solovyeva VV. Mouse tumor models for advanced cancer immunotherapy. Int. J. Mol. Sci. 2020;21(11):4118.

[2] Yu JW, Bhattacharya S, Yanamandra N, et al. Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments. PLoS One. 2018;13(11):e0206223.

[3] Wu K, Lin K, Li X, et al. Redefining tumor-associated macrophage subpopulations and functions in the tumor microenvironment. Front Immunol. 2020 Aug 4;11:1731.

[4] Shinde V, Burke KE, Chakravarty A, et al. Applications of pathology-assisted image analysis of immunohistochemistry-based biomarkers in oncology. Vet. Pathol. 2014 Jan;51(1):292-303.

[5] Wang Q, Guldner IH, Golomb SM, et al. Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor-resistant HER2-positive breast cancer. Nat. Commun. 2019 Aug 23;10(1):3817.

Tags: Syngeneic Models