Syngeneic tumor models used for discovery of immune therapeutics should have several features such as a long study duration, responsiveness to checkpoint inhibitors, high immune cell infiltration and a high homogeneity in tumor growth. Moreover, models should consider the ethical rules (3R reduce, refine, replace). At present, the standard implantation method for syngeneic tumor models is subcutaneous tumor cell inoculation. We have developed an alternative implantation method for syngeneic tumor models: inoculation into the mammary fat pad. Both implantation sides are heterotopic related to the original tumor entity except for syngeneic breast tumor cells. In addition, both tumor inoculation methods can easily be applied and monitored by calipering reducing the costs. We compared the two implantation methods with models MC38-CEA, Ct26wt, Hepa1-6, RENCA, LL-2, AB12, CloneM3, B16.F10, 4T1 and EMT-6 tumor cells in respect to growth characteristics and immune response. Intra-mammary tumor growth showed more homogeneity with higher final tumor volumes compared to the subcutaneous tumor growth. Moreover, in all tested syngeneic models, tumor ulceration was prevented by almost 100% when injecting the tumor cells into the mammary fat pad. In contrast, animals of the subcutaneous tumors were mainly euthanized due to tumor ulceration. Both findings favor the mammary fat pad injection with regard to the 3R rules by strongly reducing tumor ulceration (refinement) and animal number due to a more homogenous growth (reduction). In addition, the immune checkpoint inhibitor treatment was tested and found comparable between the intra-mammary and subcutaneous models. The presence of immune cell populations was investigated in the Ct26wt colon tumor model time-dependently by flow cytometry using a 17 marker-staining panel . The number of isolated cells per gram tumor mass was more than doubled in the intra-mammary tumors. In conclusion, tumor models of the heterotopic intra-mammary implantation side are found to be superior compared to the traditional subcutaneous tumor models: a higher tumor homogeneity with no tumor ulceration, combined with a high number of immune cells and connective stroma tissue, making the mammary fat pad implantation of syngeneic tumor cells the implantation side of choice. Citation Format: Cynthia Obodozie, Susanne Ruf, Gojko Bijelic, Sandra Moor, Bianca Giesen, Ulrike Leisegang, Sebastian Dempe, Holger Weber. Mammary fat pad injections: An alternative implantation method for syngeneic tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A012. doi:10.1158/1535-7163.TARG-19-A012

Checkpoint inhibitor treatment has become a common therapy of various cancer types. Still, there is a need for well-characterized preclinical mouse models, as clinical data indicates that patients only partially respond to immune modulatory regiments. When compared to the classical subcutaneous or subQperior࣪ (implantation into the mammary fat pad) syngeneic mouse models, orthotopic models are considered more predictive, since implantation of tumor cells into the organ of origin allows organotypical interaction between tumor cells and the surrounding stroma, including immune cells. Up until now, our standard procedure for an orthotopic Hepa1-6 liver model is the injection of Hepa1-6 cells into the spleen with the subsequent migration of the injected Hepa1 6 cells into the liver for five minutes via Vena lienalis. A major drawback of this method for a syngeneic model in immune-competent mice is the following resection of the spleen, a prominent secondary lymphoid organ. Therefore, we tested two other implantation methods: the direct injection of Hepa1-6 cells into a liver lobe using Matrigel࣪ and the injection of Hepa1-6 cells into the portal vein. In contrast to the standard procedure, both new test methods have in common an excessive bleeding potential which must be prevented. As the Hepa1-6 cells are transduced with luciferase, their growth can be monitored in vivo by bioluminescence imaging and the growth characteristics will be compared. In addition to the growth, the composition of the immune populations will be analyzed staining with the all-in-one flow cytometry panel which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. The data will help to select the most suitable method for testing new drug candidates in an orthotopic liver tumor environment. Citation Format: Cynthia Obodozie, Sandra Moor, Gojko Bijelic, Muriel Malaisé, Philipp Metzger, Holger Weber. Comparison and consequences of different implantation techniques on the orthotopic growth of syngeneic Hepa1-6 liver cancer cells [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 1621.

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