Twist1 was detected in mesenchymal cells of mammary fibroadenoma and invasive components of breast carcinoma in rats

Abstract

Fibroadenoma (FA) is a common mammary fibroepithelial tumor. The tumor size of the FA is increased by estrogen, progesterone, prolactin, and pregnancy, whereas it decreases after menopause. These observations in humans indicate that FA is hormone dependent. In rats, the most common mammary neoplasm is also FA. Expression levels of Twist1, a transcriptional regulator of epithelial-mesenchymal transition, were examined in paraffin-embedded tissue sections of an experimental rat breast model to find physiological alternations coincident with reproductive hormonal changes. Twenty-three Fischer 344/Brown Norway F1 hybrid rats were used as 14- to 16-week-old adolescent rats (n=3), pregnant rats (n=4), and lactating rats (n=6) in addition to rats over 100-weeks-old that exhibited aging (n=3) and FA (n=7). Seventy-six cases of chemically induced breast carcinoma and two cases of FA in Sprague Dawley rats were also examined. Using tissue sections, we observed that Twist1-positive mesenchymal cells were predominantly located in the periductal region in adolescent and pregnant rats and in the terminal duct lobular unit in pregnant and elderly rats. Twist1 was also expressed diffusely in the mesenchymal cells of FA rats. Twist1-positive cancer-associated mesenchymal cells were found more frequently in the invasive components of breast carcinomas than in intraductal components. The expressions of Twist1 in mesenchymal cells were induced by physiological and pathological stimuli, suggesting the biological role of Twist1 in tissue structure. Further study may reveal the role of Twist1 in mesenchymal cells of mammary glands in rats.

Keywords: Twist1; chemically induced breast carcinoma; fibroadenoma; mesenchymal cell; rat model.

Fig. 1.

Expression of Twist1 in the breast of adolescent, pregnant, lactating and elderly rats. (A) Representative images of the periductal area and terminal duct lobular unit (TDLU) are shown for adolescent, pregnant, lactating, and elderly rats (bar, 50 μm). In the periductal area, Twist1-positive mesenchymal cells predominantly emerged in the adolescent and pregnant stages. In the TDLU, Twist1-positive mesenchymal cells were observed in the pregnant and elderly stages (arrowheads, Twist1-positive cells). (B) In the periductal area, there were significant differences in the Twist1-positive ratio among the adolescent, pregnant, and elderly stages. (ANOVA, p<0.0001; *p<0.05 vs the elderly stage; **p<0.01 vs the elderly stage; ***p<0.001 vs the lactating stage). (C) In the TDLU, there were significant differences in the Twist1-positive ratio among the pregnant, elderly and adolescent stages (ANOVA, p=0.0005; *p<0.05 vs the adolescence). N.A. stands for not available due to the disappearance of mesenchymal cells during the development of lactation.

Fig. 2.

Twist1 was diffusely expressed in fibroadenoma (FA) in the mammary glands of rats. Representative images of immunohistochemistry in FA. Mesenchymal cells in FA were positive for Twist1 but were negative for myoepithelial markers (p63, α-smooth muscle actin (α-SMA), and CD10) and an epithelial marker (AE1/3) (bar, 50 μm).

Fig. 3.

Expression of Twist1 in mesenchymal cells favored invasive components of chemically induced breast carcinoma (IBC) in rats. Representative images of the intraductal component of a carcinoma and IBC are shown. α-smooth muscle actin (α-SMA), a myoepithelial marker, was visualized as dark blue; AE1/3, an epithelial marker (ductal carcinoma component), was visualized as bright blue, and Twist1 was visualized as brown. (A) There was only a limited number of Twist1-positive cancer-associated mesenchymal cells (brown) in the stroma of the intraductal component of the carcinoma, confirmed by the presence of α-SMA (dark blue). (B) There were many Twist1-positive and α-SMA negative cancer-associated mesenchymal cells (arrow heads) in the desmoplastic stroma of the IBC, confirmed by the absence of α-SMA-positive myoepithelial cells (bar, 50 μm). (C) The ratio of Twist1-positive mesenchymal cells is shown. The mesenchymal cells of the IBC expressed significantly greater number of Twist1 than the intraductal components did (ANOVA, p=0.0267; *p<0.05 vs IBC).