The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Syngeneic in vivo tumor models are valuable for the development and investigation of immune-modulating anti-cancer drugs. In the present study, we established a novel syngeneic in vivo model type named mouse-derived isografts (MDIs). Spontaneous MDIs (sMDIs) were obtained during a long-term observation period (more than one to two years) of naïve and untreated animals of various mouse strains (C3H/HeJ, CBA/J, DBA/2N, BALB/c, and C57BL/6N). Primary tumors or suspicious tissues were assessed macroscopically and re-transplanted in a PDX-like manner as small tumor pieces into sex-matched syngeneic animals. Nine outgrowing primary tumors were histologically characterized either as adenocarcinomas, histiocytic carcinomas, or lymphomas. Growth of the tumor pieces after re-transplantation displayed model heterogeneity. The adenocarcinoma sMDI model JA-0009 was further characterized by flow cytometry, RNA-sequencing, and efficacy studies. M2 macrophages were found to be the main tumor infiltrating leukocyte population, whereas only a few T cells were observed. JA-0009 showed limited sensitivity when treated with antibodies against inhibitory checkpoint molecules (anti-mPD-1 and anti-mCTLA-4), but high sensitivity to gemcitabine treatment. The generated sMDI are spontaneously occurring tumors of low passage number, propagated as tissue pieces in mice without any tissue culturing, and thus conserving the original tumor characteristics and intratumoral immune cell populations.

In this second study, we established syngeneic in vivo models named carcinogen-induced mouse-derived isografts (cMDIs). Carcinogen-induced tumors were obtained during short-term observation (3–9 months) of CBA/J mice treated with various administration routes with 3-methylcholanthrene (MCA) or N-methyl-N-nitrosourea (MNU) as carcinogens. During necropsy, primary tumors and suspicious tissues were assessed macroscopically and re-transplanted (in PDX-like manner) into sex-matched syngeneic animals. Outgrowing tumors were histologically characterized as either spinocellular carcinoma (1/8) or various differentiated sarcomas (7/8). Growth curves of four sarcomas showed striking heterogeneity. These cMDIs were further characterized by flow cytometry, RNA sequencing, or efficacy studies. A variable invasion of immune cells into the tumors, as well as varying expression of tyrosine kinase receptor, IFN-γ signature, or immune cell population marker genes could be observed. Immune checkpoint inhibitor treatment (anti-mPD-1, anti-mCTLA-4, or a combination thereof) showed different responses in the various cMDI models. In general, cMDI models are carcinogen-induced tumors of low passage number that were propagated as tissue pieces in mice without any tissue culturing. Therefore, the tumors contained conserved tumor characteristics and intratumoral immune cell populations. In contrast to the previously described spontaneous MDI, carcinogen induction resulted in a greater number of individual but histologically related tumors, which were preferentially sarcomas.

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