Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

Mammary glands undergo morphologic and biochemical changes during various physiologic stages of life, specifically during the transition from virgin to pregnancy, lactation, and involution (1–3). The complete cycle of structural and functional differentiation depends on the coordinated action of prolactin, insulin, adrenal corticoids, and ovarian hormones (4,5). Atypical ductal hyperplasia is an abnormal ductal epithelial cell proliferative condition that does not invade the periductal stroma (6). In women, atypical ductal hyperplasia, which may become more aggressive and ultimately fill the lumen of the duct, is considered to be a physiologic precursor to the development of ductal carcinoma in situ (DCIS). Although the histopathology of DCIS subtypes is well defined, there are few experimental models to evaluate the molecular mechanisms underlying DCIS formation or to evaluate cancer chemopreventive agents. One model is the mouse mammary gland organ culture (MMOC) (7,8), in which the entire cycle of mammary gland morphology and physiology can be simulated with appropriate hormonal supplementation of a chemically defined medium. We have shown previously that the MMOC model is useful for evaluating the underlying molecular mechanisms of lesion formation because mammary glands exposed to 7,12-dimethylbenz[a]anthracene (DMBA) develop hyperplastic mammary alveolar lesions in the presence of the nonovarian steroid hormones aldosterone and hydrocortisone (9). These lesions do not regress to the nonproliferating state comprised mainly of ducts and a few end-buds after the removal of growth-promoting hormones. Moreover, cells isolated from these lesions form adenocarcinomas when transplanted into syngeneic mice (10). In addition, the model is useful for testing the efficacy of chemopreventive agents to inhibit DMBA-induced lesions (11,12), although tamoxifen did not reduce lesion formation. Because of the proposed role of ovarian hormones in breast carcinogenesis, we evaluated the effects of estrogen and progesterone on DMBA-induced lesions in the MMOC model. Ductal lesions were induced by incubating mouse mammary glands in serum-free Waymouth MB752/1 medium (7,8), in which aldosterone and hydrocortisone were replaced with estradiol17 (0.001 g/mL) and progesterone (1 g/mL). The glands were treated with DMBA (2 g/mL) for 24 hours on the third day of culture. After 24 days, glands were fixed in 10% formalin, and histopathologic sections were evaluated for ductal lesions. For progesterone receptor analyses, mammary glands were incubated with growth-promoting hormones either alone or in the presence of estradiol-17 (1 nM) and/or tamoxifen (1 M) for 6 days. On day 6, the glands were fixed in 10% formalin, and histologic sections were prepared and immunostained with antibodies to the progesterone receptor (Neomarkers, Fremont, CA) (13). Sections were evaluated semiquantitatively for the expression of progesterone receptor according to the intensity of staining (14). The difference between means of percent incidence (of atypical ductal hyperplasia) and percent induction (of progesterone receptor expression) of control and treated groups was analyzed by use of Student’s t test for independent samples (SPSS® statistical software; version 6.1.3; Chicago, IL). All statistical tests were twosided. Histologically, ovarian hormonedependent lesions induced by DMBA in MMOC were predominantly of ductal rather than alveolar origin, similar to human breast hyperplastic and premalignant lesions. The ducts in control glands (i.e., not treated with DMBA) were largely lumina lined with one or two layers of epithelial cells (Fig. 1, A). The ducts in DMBA-treated glands were thickened and lined with five to six layers of hyperplastic cells (defined by a thickness of more than three cell layers) (Fig. 1, C). The lumina of some ducts were occluded completely by intraductal outgrowths, and the epithelial cells often formed alveolar and papillary structures (Fig. 1, E). Transverse sections through the lesions showed a combination of proliferating epithelial cells and areas of necrosis (Fig. 1, F). Close examination of the intraductal lesions showed that aggressive lesions (i.e., those completely occluded with ductal epithelial cells) were composed of atypical cells (variable in size and form), nuclei with intense chromatin staining, reduced intracellular spaces, and a reduced number of mitotic figures (Fig. 1, G). Light microscopic images of the sectioned glands were converted to digitized images by use of software written in MATLAB® (Source: MATLAB version 5.3 licensed to the Department of Electrical Engineering and Computer Science, University of Illinois, Chicago) to quantify the ductal lesions. The score for the area covered by epithelial cells was 0.05 for control glands (Fig. 1, B), 0.72 for hyperplastic lesions (Fig. 1, D), and 1.13 for aggressive ductal lesions (image not shown). Various degrees (ranging from greater than three layers of epithelial cells to completely occluded ducts) of intraductal atypical hyperplastic lesions were found in 387 of 486 fields from histologic sections of 61 DMBA-treated glands, which was equivalent to an incidence of 76.1% (95% confidence interval [CI] 69.3% to 82.9%). There were a few falsepositive lesions in four of 74 fields from sections of seven control glands, which was equivalent to an incidence of 5.4% (95% CI 2.8% to 8%). There was a