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Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

Rajendra G. Mehta, Krishna P. L. Bhat, Michael E. Hawthorne, Levy Kopelovich, Rajeshwari R. Mehta, Konstantin Christov, Gary J. Kelloff, Vernon E. Steele, John M. Pezzuto

Affiliations of authors: R. G. Mehta, M. E. Hawthorne, R. R. Mehta, K. Christov (Department of Surgical Oncology, College of Medicine), K. P. L. Bhat, J. M. Pezzuto (Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy), University of Illinois at Chicago; L. Kopelovich, G. J. Kelloff, V. E. Steele, Chemopreventive Agent Development Group, Division of Cancer Prevention and Control, National Cancer Institute, Bethesda, MD.

Correspondence to: John M. Pezzuto, Ph.D., Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (M/C 877), University of Illinois at Chicago, 833 South Wood St., Chicago, IL 60612 (e-mail: jpezzuto{at}uic.edu).

Mammary glands undergo morphologic and biochemical changes during various physiologic stages of life, specifically during the transition from virgin to pregnancy, lactation, and involution (13). 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 estradiol-17{beta} (0.001 µg/mL) and progesterone (1 µg/mL). The glands were treated with DMBA (2 µg/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{beta} (1 nM) and/or tamoxifen (1 µM) 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 two-sided.

Histologically, ovarian hormone-dependent 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. 1Go, 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. 1Go, C). The lumina of some ducts were occluded completely by intraductal outgrowths, and the epithelial cells often formed alveolar and papillary structures (Fig. 1Go, E). Transverse sections through the lesions showed a combination of proliferating epithelial cells and areas of necrosis (Fig. 1Go, 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. 1Go, G).



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Fig. 1. Histopathologic characterization of mammary ductal lesions. Mammary glands were incubated with insulin (5 µg/mL), prolactin (5 µg/mL), estradiol-17{beta} (0.001 µg/mL), and progesterone (1 µg/mL) for the first 10 days in culture, followed by withdrawal of all hormones except insulin and incubation for an additional 14 days. 7,12-Dimethylbenz[a]anthracene (DMBA) (2 µg/mL) was added for 24 hours on day 3 of culture. After a total of 24 days in culture, glands were fixed in formalin, processed, and stained with hematoxylin–eosin for histopathologic evaluation. Panel A: Control glands treated only with the vehicle (dimethyl sulfoxide) for the first 10 days show one to three layers of epithelial cells lining the ducts. Panel C: Glands treated with DMBA show hyperplastic lesions, as evidenced by several layers of epithelial cells, with a narrowing of the lumen. Panel E: Glands treated with DMBA also exhibit atypical ductal hyperplasia, as evidenced by the duct that is completely occluded with atypical epithelial cells. Panels B and D: digitized images of panels A and C, respectively. Quantitative analysis of ductal lesions was carried out by use of the image analysis software written in MATLAB® (Source: MATLAB version 5.3 licensed to the Department of Electrical Engineering and Computer Science, University of Illinois, Chicago). The epithelial cells from the control non-DMBA-treated glands were used as negative controls. Panel F: transverse section of a completely occluded duct containing atypical hyperplastic epithelial cells. Arrow indicates area of necrosis. Panel G: atypical ductal epithelial cells with varying cellular and nuclei size and shape. Panel H: Glands incubated with 1 µM tamoxifen for the first 10 days of culture exhibit normal mammary ductal structures. Panels I and J: For the immunohistochemistry of progesterone receptor expression in mammary glands, glands were incubated with insulin and prolactin for 6 days, histologic preparations were made, and the sections were then immunostained for the progesterone receptor (13). To block nonspecific antibody reactions, the sections were treated with 5% dried skim milk for 10 minutes and then incubated with primary antibody against progesterone receptor overnight at 0 °C–4 °C, according to the manufacturer's recommendations (Neomarkers, Fremont, CA). The sections were incubated with biotinylated anti-rabbit anti-mouse-linked antibody (Dako Corp., Carpinteria, CA) for 10 minutes followed by a 10-minute incubation with peroxidase-conjugated streptavidin (Dako Corp.) and AEC chromogen (BioGenex Laboratories, San Ramon, CA) in H2O2 substrate for 5 minutes. The sections were evaluated semiquantitatively for the expression of progesterone receptor according to the intensity of staining (14). The cells were scored as follows: - = nonstaining; + = observable staining; ++ = marked staining; and +++ = intense staining. The percent intensity was calculated by use of the formula: intensity (%) = 100*(n+ + n++ + n+++)/(n- + n+ + n++ + n+++). Panel I: Glands treated with estradiol-17{beta} (1 nM) show intense progesterone receptor expression. Panel J: Glands treated with estradiol-17{beta} (1 nM) and tamoxifen (1 µM) for 6 days show reduced progesterone receptor expression compared with estradiol-17{beta}-treated glands shown in panel I. Original magnification for all panels, x40.

 
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. 1Go, B), 0.72 for hyperplastic lesions (Fig. 1Go, 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 false-positive 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 statistically significant difference in the occurrence of atypical ductal hyperplasia (95% CI for the mean difference = 55.7% to 96.1%; P<.001) between the DMBA-treated and control glands.

We next evaluated the effect of tamoxifen and other modulators of estrogen function on DMBA-induced atypical ductal hyperplasia. Vehicle (dimethyl sulfoxide)-treated control glands had normal morphology. Glands treated with tamoxifen (1 µM) during the first 10 days of growth, in the presence of estrogen and progesterone but in the absence of DMBA, had thin ducts and small normal end-buds. DMBA-treated glands had few alveoli, thickened, enlarged end-buds, and dense hyperplastic ducts (Fig. 1Go, C and E). The number of hyperplastic ductal lesions was greatly reduced in glands treated with DMBA in the presence of tamoxifen (Fig. 1Go, H). Additional antiestrogens and modulators of estrogen metabolism were also found to inhibit DMBA-induced ductal lesions (IC50 [i.e., concentration of test substance required to reduce ductal hyperplasia by 50%] values are summarized in Table 1Go).


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Table 1. Effect of modulators of estrogen function on 7,12-dimethylbenz[a]anthracene-induced mammary ductal lesions in organ culture*
 
We next assessed the estrogenic action in MMOC by immunohistochemically examining progesterone receptor expression. Mammary glands were treated for 6 days with insulin (5 µg/mL)- and prolactin (5 µg/mL)-containing medium with estradiol-17{beta} (1 nM) or with estradiol-17{beta} (1 nM) plus tamoxifen (1 µM) and then fixed, processed, sectioned, and immunostained as described previously (13). Glands grown in the absence of estradiol-17{beta} expressed the progesterone receptor at a basal level, with an intensity score (14) of 24.4% (95% CI = 17.4% to 31.2%). Glands grown in the presence of estradiol-17{beta} (Fig. 1Go, I) expressed higher levels of the progesterone receptor, with an intensity score of 78.3% (95% CI = 60.3% to 95.4%) and a statistically significant mean difference compared with control glands (95% CI for the mean difference = 36% to 71.1%; P<.001). Tamoxifen-treated glands expressed lower levels of the progesterone receptor (Fig. 1Go, J), with an intensity score of 30.3% (95% CI = 23.6% to 36.2%) and a statistically significant mean difference compared with estradiol-17{beta}-treated glands (95% CI for the mean difference = 30% to 65%; P<.001). These results indicate that the induction of the progesterone receptor can be used as a marker for estrogenic activity in MMOCs.

One of the initial effects of estradiol-17{beta} in mammary cell differentiation is the induction of estrogen-inducible genes, including the progesterone receptor (15). We found that the expression of progesterone receptors was localized in the epithelial cell nuclei of glands incubated with insulin, prolactin, and estradiol-17{beta} and that treatment of glands with tamoxifen for 6 days decreased estrogen-inducible progesterone receptor expression. Consistent with our studies, a recent report (16) showed that progesterone receptor knockout mice developed fewer DMBA-induced tumors than isogeneic wild-type mice. These data indirectly suggest a regulatory role for progesterone and the progesterone receptor in carcinogenesis. Although the complete role of the progesterone receptor in the development of breast cancer remains unclear, the induction of DMBA-induced lesions will be a valuable tool for studying the mechanism of progesterone action in mammary carcinogenesis.

In this study, we show for the first time that DMBA can induce estrogen- and progesterone-dependent mammary atypical ductal hyperplastic lesions in MMOC with high frequency. Although histologically distinct from human DCIS, these estrogen-dependent lesions can be considered analogous to atypical ductal hyperplasia observed in women, which are distinct and often a prerequisite to the development of breast can-cer (6). Therefore, our MMOC model could serve as a tool for identifying and studying the alterations in molecular markers during the development of atypical ductal lesions and DCIS. Moreover, because tamoxifen and other modulators of estrogen action suppressed the formation of estrogen-dependent ductal lesions (Table 1Go), this MMOC model is useful for determining the potential of cancer chemopreventive agents to inhibit the development of transformed atypical hyperplastic ductal lesions.

NOTES

Supported by Public Health Service contracts CN55135 and CN65114 and by grant P01CA48112 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

We thank Drs. Henry Thompson and Meenakshi Singh, AMC Cancer Research Center, Denver, CO, for valuable suggestions during the initiation of the project and critical evaluation of the manuscript.

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Manuscript received November 20, 2000; revised April 24, 2001; accepted May 4, 2001.


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