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