Utilization of animal models with an intact immune system is critical for the evaluation of immune-based therapeutic strategies and vaccine development. An SV40 TAg transgenic model of prostate cancer [55
] has been used to study the effects of combining blockade of cytotoxic T lymphocyte antigen 4 (CTLA-4) and vaccination with granulocyte macrophage colony stimulating factor (GM-CSF;Gvax) and subsequent derivatives of this vaccine strategy [56
]. The C57BL/6 syngeneic mouse ovarian cancer model developed by Roby et al, [10
] has been used for studies of the contribution of cells in the tumor microenvironment, including epithelial-stromal cell interactions, VEGF induced-effects on tumor vasculature and tumor cell-secreted factors that stimulate cytokine production, macrophage infiltration and vascularization that favor tumor growth and progression [14
]. Similar studies would be difficult to impossible to conduct in immunodeficient mice. The availability of an additional syngeneic mouse model of EOC will allow cross-comparison of mouse models and validation of key findings.
The functional utility of animal models of human cancer depends largely on the extent to which the animal model recapitulates the histology and biological behavior of the disease in humans. Many transgenic tumor models have been developed using the immediate early region of the SV40 virus containing the potently oncogenic large and small T antigen (TAg
) genes [55
]. The continued utility of SV40 TAg models in studying cancer is underscored by seminal contributions to our understanding of the "angiogenic switch" [67
] and tumor progression and invasion [72
]. Importantly, a recent study [73
] identified an integrated gene expression signature from three distinct TAg mouse models (i.e., mammary, prostate and lung cancer models) that is comparable to a signature associated with the aggressive biological behavior and prognosis for several human epithelial tumors, including breast cancers. Results from this study showed that tumors arising in TAg-based mouse models share common features of gene expression with human cancer and are relevant preclinical models [73
Female transgenic C57BL/6 TgMISIIR-TAg-DR26
mice develop spontaneous bilateral ovarian carcinoma with 100% penetrance [28
]. Tumor progression in these mice is characterized by widespread peritoneal dissemination and the development of malignant ascites and tumor morphology and histology of the tumors closely resembles high-grade serous adenocarcinomas, the most common histologic subtype of EOC detected in women. Tumors and cell lines derived from primary tumors and ascites of tumor bearing mice exhibit several characteristics in common with human EOC cell lines and tumors including AKT/mTOR activation, COX1 overexpression and VEGF overexpression and secretion ([28
] and the present study). In addition, a verapimil-sensitive Hoescht dye-excluding ovarian carcinoma side population (SP), a potential population of ovarian cancer initiating cells, was identified in MOVCAR cell lines [48
]. Ovarian tumors arising in C57BL/6 TgMISIIR-TAg-DR26
mice are sensitive to standard combination platinum and taxane chemotherapy and to mTOR inhibition with Everolimus (RAD001) [28
]. These observations underscore the potential utility of these transgenic mice for preclinical evaluation of therapeutic agents. However, reflecting its relation to the biology of human EOC, tumor formation in this transgenic model is also stochastic, resulting in variation in the latency of tumor formation and time to metastasis. This necessitates relatively large cohorts of mice and non-invasive longitudinal in vivo
imaging such as MRI to optimize results of therapeutics studies.
To overcome the limitations encountered with spontaneous tumor development, we isolated individual transgenic lines of non-tumor prone C57BL/6 TgMISIIR-TAg
transgenic mice that can serve as syngeneic immunocompetent hosts for allografted TAg expressing MOVCAR cells isolated from tumor bearing C57BL/6 TgMISIIR-TAg-DR26
mice. Syngeneic mouse models of EOC in which spontaneously transformed ID-8 MOSEC grown as allografts in C57BL/6 recipients [10
] or HM-1 tumor cells grown as allografts in B6C3F1 recipients [74
] have been previously described. These syngeneic models have been used successfully for preclinical evaluation of therapeutic agents and studies of the role of the tumor microenvironment on ovarian tumor growth and progression [11
]; however, these models each rely on single mouse ovarian carcinoma cell lines in which the underlying molecular mechanisms of malignant transformation remain undefined.
The ease of establishment of TAg-transformed MOVCAR cell lines in culture has enabled the isolation of a large number of distinct cell lines, several of which are described in the present study. Although derived from an inbred strain of mice, the stochastic manner in which tumors arise in C57BL/6 TgMISIIR-TAg-DR26 mice results in intrinsic differences in MOVCAR cell lines derived from individual tumor-bearing mice. MOVCAR cell lines grown in culture exhibit different growth rates and expression of proteins associated with EOC, such as levels of secreted VEGF. These cell lines also exhibit differences when grown in vivo. For example, some cells lead to very rapid growth and production of voluminous malignant ascites, whereas other cells are slower growing and produce less ascites. Interestingly, the cell lines that result in the highest levels of ascites production in vivo are the cell lines that exhibit the highest levels of VEGF secretion in vitro. These observations suggest that although the primary oncogenic stimulus driving tumorigenesis in C57BL/6 TgMISIIR-TAg-DR26 transgenic mice is the same in all animals, there are likely additional genetic, epigenetic and/or gene expression alterations that contribute to ovarian tumor progression, and identification of these alterations may contribute to our understanding of human EOC. Moreover, once identified, the role of specific alterations in gene function in ovarian tumorigenesis can be studied in these cell lines as they are readily amenable to direct manipulation using established strategies for ectopic gene expression or RNA interference.
With regard to preclinical evaluation of novel therapeutic agents, our syngeneic mouse model of EOC provides several advantages. First, tumors are grown in fully immunocompetent mice enabling the evaluation of vaccine and immune-based therapeutic strategies. Second, TgMISIIR-TAg-Low transgenic mice have been fully backcrossed to a pure C57BL/6 genetic background, exhibit normal fertility and lifespan and do not develop tumors. Thus, large cohorts of mice can be established for synchronous allograft initiation without interference of tumor growth initiated from the host. Third, the availability of multiple distinct MOVCAR cells lines for evaluation avoids issues of cell line-specific effects, and because MOVCAR cells are easily manipulated in culture, on-target effects of therapeutics can be confirmed in parallel using RNAi based strategies for direct target knockdown. Finally, the ability to easily express reporter genes in MOVCAR cells facilitates strategies for non-invasive in vivo optical imaging such as bioluminescent, fluorescent and near infrared fluorescent imaging.