Abstract 1657: Syngeneic tumor development, treatment responses and immune infiltrate in subcutaneous and orthotopic mouse models
Authors: Cold Sigrid , AL-Haidar Saif , Joergensen Jennifer Solgaard , Engel Trine Bjoernbo , Fliedner Frederikke Petrine , Kristensen Lotte Kellemann , Kjaer Andreas , Nielsen Carsten Haagen 
Published: June 2022

Abstract
Background: Syngeneic mouse tumor models are widely used and effective tools to evaluate efficacy of novel anti-cancer therapies. They are often implanted subcutaneously in a microenvironment that is unrelated to the site of origin. This can be problematic when evaluating immune modulating therapies as the tumor microenvironment is known to differ substantially between orthotopic and subcutaneous models. Here, we report the development and characterization of several syngeneic mouse tumor models implanted subcutaneously and orthotopically. The models are characterized by immune infiltrate and response to novel and conventional therapies.
Methods: Murine MC38 (colon), TRAMPC1 (prostate) and GL261 (glioma) cell lines were implanted subcutaneously or orthotopically in immune competent mice. Tumor growth was monitored at multiple timepoints by external caliper measurements, magnetic resonance (MR) or ultrasound imaging. At confirmed tumor take, mice were treated with external radiation therapy (RT) or immune checkpoint inhibitors. Single cell suspensions were prepared from tumors and tumor draining lymph nodes (TdLNs) at various timepoints after treatment initiation and immuno-profiling was evaluated by multicolor flow cytometry. The tissues were analyzed for proportions of the following cell populations; CD4+ and CD8+ T lymphocytes, myeloid derived suppressor cells (MDSCs), NK cells and macrophages.
Results: Subcutaneous and orthotopic tumor volumes developed at a similar pace. However, orthotopic tumor volumes estimated by MR imaging were detectable at earlier timepoints compared to subcutaneous volumes estimated by caliper measurements. Response to therapy was dependent on implantation site. This was further highlighted by the immune infiltration analysis, where CD4+ and CD8+ tumor infiltrating lymphocytes differed markedly between subcutaneous and orthotopic tumors. Also, the temporal profile of tumor infiltrating immune cells up to 14 days following therapy was established in the models.
Conclusion: Subcutaneous and orthotopic versions of syngeneic mouse tumor models were successfully established and characterized with respect to their tumor immune microenvironment. The models displayed varying sensitivity towards RT and immune checkpoint inhibitors. Response to therapy was furthermore dependent on implantation site. Altogether, these advanced models are powerful tools for investigating treatment effect within preclinical immuno-oncology.
Citation Format: Sigrid Cold, Saif AL-Haidar, Jennifer Solgaard Joergensen, Trine Bjoernbo Engel, Frederikke Petrine Fliedner, Lotte Kellemann Kristensen, Andreas Kjaer, Carsten Haagen Nielsen. Syngeneic tumor development, treatment responses and immune infiltrate in subcutaneous and orthotopic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1657.