Checkpoint inhibitor treatment has already become a common therapy of various cancer types. Still there is a growing need for well-characterized preclinical mouse models, as clinical data indicate that patients only partially respond to this regiment. We examined the efficacy of anti-CTLA-4, anti-PD-1 and anti-PD-L1 therapy on 4T1, B16.F10, Clone M-3, CT26wt, LL/2, MC38-CEA and RENCA syngeneic tumor models. The outcome demonstrates a large variation in the response to the immune checkpoint therapy among the analyzed tumor models. Poorly immunogenic models like the LL/2 tumor did not respond to any given therapy, whereas highly immunogenic tumor models like CT26wt or MC38-CEA tumors were inhibited by all tested immunotherapies. The CT26wt tumor model was further characterized in a re-challenge experiment. Growth retardation was observed in CT26wt-tumor bearing mice treated with anti-PD-L1 antibody. A fraction of mice responded with a complete regression of the tumor. These mice were repeatedly challenged subcutaneously with a high number of fresh CT26wt tumor cells. No re-growth of CT26 tumors was observed, whereas challenging with fresh 4T1 tumor cells resulted in a rapid 4T1 growth in these mice, demonstrating a specific immune protection against CT26wt tumors. In addition all syngeneic tumor models were analyzed for the distribution of immune cells such as Treg cells, M1/M2 macrophages and M/PMN-MDSCs in tumor tissue based on multi-color flow-cytometric analysis. These data may help to select a suitable model for testing new drug candidates and define a sensitive combination therapy to support the anti-tumor immune defense in addition to checkpoint inhibitor treatment. Citation Format: Peter Jantscheff, Cynthia Schaefer-Obodozie, Sandra Moor, Holger Weber. Anti-PD-1, Anti-PD-L1 and Anti-CTLA-4 checkpoint inhibitor treatment leads to different responses in syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3216.

Many human breast cancer cell lines have been implanted into immunodeficient mice in order to establish in vivo xenograft models, subcutaneous or orthotopic variants, in order to mimic this type of cancer as good as possible. However, especially the metastasising capabilities of these models turned out to be highly disappointing. In order to get more reliable models, breast cancer cells have often been implanted intravenously or intracardially rather than orthotopically. These models do work fine but resemble only limited parts of the complicated metastasis process. Therefore, a metastasis event originated from an orthotopically growing breast tumor seems more appropriate and displays an expanded view on the process of metastasis. In the model presented here, the murine brast cancer cell line 4T1 was transduced with fire fly luciferase and implanted orthotopically into the fat pad of female BALB/C mice. The metastasis rate in this model is known to be better than in the common human xenografts transplanted in mice, although still limited. In order to get higher metastasis rates we tried to improve this model by resecting and recultivating the metastases of the invaded lungs after orthotopic implantation of the 4T1 cells. Cultured cells were checked for in vitro luciferase activity in parallel with ex vivo analysis of the lung tissue part which was used for the isolation of the cells. After using this approach for several rounds, what we refer to as “subpopulationing”, we checked for increased metastasis properties of this newly isolated breast cancer cells. The new 4T1-M-ortho-luc cells were implanted ortotopically in parallel to the parental 4T1-luc cell line and we analysed 9 different organs ex vivo for luciferase activity. Whereas in the parental model only in lung tissue metastasis in all implanted mice (12/12) was detectable, the metastasis rate in the new model, generated by “subpopulationing”, bounced up to 4 organs with measurable metastasis in 100 % of the mice and in general to a much higher degree of metastasis in most other organs. Only in liver tissue no metastases could be detected in both models. Treatment of the mice with Doxorubicin clearly diminished tumor growth in both breast cancer models and can therefore serve as a potent positive control here. With this new 4T1-M-ortho cell line we have available a metastasing breast cancer cell line that shows reliable metastasis observed in several organs of the mouse when implanted orthotopically. This model is suitable for testing potential anti-meatstasizing compounds aginst breast cancer. In addition, since this model is established in immunocompetent mice, and is therefore also perfectly suited for approaches involving the immune system of breast cancer patients. Citation Format: Andreas Lingnau, Steffen Hoffmann, Sandra Moor, Cynthia Schaefer-Obodozie, Christoph Schaechtele. Establishment of a reliable metastasizing syngeneic breast cancer mouse model using orthotopically implanted 4T1 cells after several rounds of isolating and reimplanting lung metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1222. doi:10.1158/1538-7445.AM2014-1222

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.