Abstract 3275: Development of a new ex-vivo orthotopic model-Human breast cancer cells in mouse mammary gland organ culture

Abstract

Abstract Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogen-induced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC)). Three to four week old female BALB/c mice were sensitized with estradiol (1μg) + progesterone (1mg) for 9 days. On the 10th day animals were sacrificed and 2.5x104 T47Dparental or T47D aromatase overexpressing cells were injected into the fourth pair of thoracic mammary glands. The glands were excised then cultured at 37°C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS/supplemented with Testosterone (1nM) and progesterone (1uM) and growth promoting hormones (5 µg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific Ck-18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex-vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3275. doi:1538-7445.AM2012-3275