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

Prostate cancer is one of the most frequently diagnosed cancer entities in men. It can be estimated that in 80% of all men reaching the age of 80 years prostate tumors are present. Since a depletion of testosterone in prostate cancer therapies is often performed, androgen-dependent and -independent prostate carcinoma cell lines have been widely used to establish mouse in vivo xenograft models in order to identify novel drugs helping to eliminate prostate tumors. A commercially available LNCaP cell line was shown to be androgen-dependent since addition of synthetic androgens to androgen-free culture medium was able to restore proper cell proliferation in cell culture. However, when implanted in SCID mice in order to establish a xenograft model, the take rate turned out to be very low and tumor growth was highly heterogeneously. Therefore, we resected and recultivated one of the few well-growing tumors and reimplanted the arising subpopulation subcutaneously in mice. After performing two rounds of this “subpopulationing” procedure we could generate a new cell line, LNCaP-(Z2), which was able to form tumors in mice with a take rate of nearly 100%, but still shows heterogenous tumor growth similar to other subcutaneously implanted prostate cancer cells (e.g. PC-3). In order to demonstrate an androgen dependency of our new LNCaP subpopulation model in vivo we used castrated mice and implanted them subcutaneously in the absence or presence of also subcutaneously inoculated testosterone pellets. For comparison PC-3 prostate cancer cells were also implanted subcutaeously in the presence or absence of testosterone. Whereas no influence of testosterone was detectable on the growth behavior of subcutaneous PC-3 xenograft tumors, the development of the LNCaP-(Z2) xenografts was strictly dependent on testosterone release. We then implanted fire fly luciferase expressing LNCaP-(Z2)-luc cells orthotopically into the prostate of castrated male SCID beige mice with or without testosterone supplementation to show testosterone-dependency. Finally, treatment of Bicalutamide, a typical member of the class of anti-androgens, which is frequently used in the treatment of prostate cancer, led to significant inhibition of tumor growth using our improved subpopulation LNCaP-(Z2) in vivo model. Bicalutmid and Flutamid, another anti-androgen agent, were used for treatment in the orthopical in vivo model. Both compounds showed a noticeable anti-tumoral effect at necropsy. Thus, we could demonstrate the model's potency as a testosterone-dependent in vivo tool suitable for screening and development of novel anti-prostate cancer drugs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B14. Citation Format: Andreas Lingnau, Sandra Moor, Steffen Hoffmann, Cynthia Schaefer-Obodozie, Ulrike Leisegang, Klotzbuecher Andrea, Schaechtele Christoph. Characterization of an in vivo generated subpopulation of human LNCaP prostate cancer cells as an improved testosterone-dependent subcutaneous as well as orthotopic in vivo mouse xenograft model for compound testing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B14.

Prostate cancer is one of the most frequently diagnosed cancer entities in men. It can be estimated that in 80% of all men reaching the age of 80 years prostate tumors are present. Since a depletion of testosterone in prostate cancer therapies is often performed, androgen-dependent and -independent prostate carcinoma cell lines have been widely used to establish mouse in vivo xenograft models in order to identify novel drugs helping to eliminate prostate tumors. A commercially available LnCaP cell line was shown to be androgen-dependent since addition of synthetic androgens to androgen-free culture medium was able to restore proper cell proliferation in cell culture. However, when implanted in SCID mice in order to establish a xenograft model, the take rate turned out to be very low and tumor growth was highly heterogeneously. Therefore, we resected and recultivated one of the few well-growing tumors and reimplanted the arising subpopulation subcutaneously in mice. After performing two rounds of this “subpopulationing” procedure we could generate a new cell line, LnCaP-Z2, which was able to form tumors in mice with a take rate of nearly 100%, but still shows heterogenous tumor growth similar to other subcutaneously implanted prostate cancer cells (e.g. PC-3). In order to demonstrate an androgen dependency of our new LnCaP subpopulation model in vivo we used castrated mice and implanted them subcutaneously with two different prostate cancer cell lines in the absence or presence of also subcutaneously inoculated testosterone pellets. Whereas no influence of testosterone was detectable on the growth behavior of subcutaneous PC-3 xenograft tumors, the development of the LnCaP-Z2 xenografts was strictly dependent on testosterone release. Finally, treatment of Bicalutamide, a typical member of the class of anti-androgens, which is frequently used in the treatment of prostate cancer, led to significant inhibition of tumor growth using our improved subpopulation LnCaP-Z2 in vivo model. Thus, we could demonstrate the model's potency as a testosterone-dependent in vivo tool suitable for screening and development of novel anti-prostate cancer drugs. Citation Format: Andreas Lingnau, Steffen Hoffmann, Cynthia Schaefer-Obodozie, Ulrike Leisegang, Andreas Klotzbuecher, Christoph Schaechtele. Characterization of an in vivo generated subpopulation of human LnCaP prostate cancer cells as an improved testosterone-dependent in vivo mouse xenograft model for compound testing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2013-2788

In drug development, predicting in vivo inhibitory drug activity from cellular assay data is difficult due to the complexity of the living system, resulting in the sacrifice of many mice in vain. In order to prescreen compounds for their in vivo activity more efficiently, the hollow fiber assay (HFA) was developed in 1995 by Hollingshead et al. The HFA assay allows the simultaneous evaluation of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers within a single mouse. We herein report on the advancement of this technology by use of luciferase-labeled tumor cell lines. The limits of detection are boosted and disturbing (non-labeled) stroma cells attached to the fibers are excluded enabling a clear separation of drug effects on the tumor cells. The effect of Gemcitabine and Paclitaxel on growth of luciferase-labeled MDA-MB-231 and A549 cells in HFA was analyzed by MTT in comparison to luciferase activity analysis. The general findings between both read-outs were comparable. The growth of MDA-MB-231 cells was inhibited by both, Paclitaxel and Gemcitabine, whereas inhibition of A549 cell growth was only observed under Paclitaxel treatment. T/C calculation for the MTT analysis predicted a partial response in all cases. In contrast, the luciferase activity analysis clearly distinguished between a complete response for MDA-MB-231 cell growth when treated with Paclitaxel and Gemcitabine and a partial response for Paclitaxel on A549 cell growth. These results correspond to the in vivo xenograft studies: T/C for Gemcitabine using MDA-MB-231 cells was 2-fold lower (16.2%) than T/C in A549 tumors using Paclitaxel (36.3%). In addition, results are shown applying this technology for up to 42 days instead of the typical 14 days, enhancing the possibility to analyze slow-acting drugs. In summary, the HFA assay for in vivo drug screening with luciferase-labeled tumor cells remarkably increases the power and predictability for drug efficacy in xenograft studies. Citation Format: Cynthia Schaefer-Obodozie, Ulrike Leisegang, Susanne Ruf, Bianca Giesen, Christoph Schaechtele, Holger Weber. Boosting of in vivo hollow fiber assay by using luciferase-labeled tumor cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A16.

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