Long-term survival of adult mouse mammary glands in culture and their response to a retinoid.

Previous studies have shown that the development of mam mary alveolar lesions (MALs) precedes the emergence of overt mammary tumors in high-breast-cancer strains of mice, such as Rill, that are infected with exogenous murine mammary tumor virus. We cultured MAL-containing whole mammary glands from 7-month-old nulliparous Rill mice for 8 days (Days 0 to 8) in a serum-free medium that contained insulin and a combination of lactogenic hormones, prolactin, aldosterone, and hydrocortisone. On the ninth day, the cultures were washed, and the glands were maintained in a medium containing insulin alone for the next 14 days (Days 9 to 23). Lobuloalveolar structures (LAs) were found to develop in the cultured glands during the 8-day period but, during the next 14 days, in the absence of lactogenic hormones, the LAs that developed during the first 8 days of culture were found to regress. However, the MALs that were present in the mammary glands before culturing remained unal tered. These results indicate that the survival of MALs is not dependent upon the lactogenic hormones but that the mainte nance of LA requires these hormones. Organ cultures of MALfree mammary glands from C57BL mice (a low-breast-cancer strain that lacks exogenous murine mammary tumor virus) main tained in the presence of the lactogenic hormones were found to develop LAs similar to those observed in the Rill cultures. Subsequent cultivation of C57BL glands in lactogenic hormonefree medium exhibited regression of LA as in the Rill cultures, and these cultured glands were found to contain a few MAL-like structures. In an attempt to evaluate the effects of retinoids on MALs, Rill mammary gland organ cultures containing MALs that had developed in vivo were incubated for the initial 8 days (Days 0 to 8) in the presence of lactogenic hormones and were then continuously exposed to 5 to 10 MM A/-(4-hydroxyphenyl)retinamide (HPR) in lactogenic hormone-free medium for the next 14 days (Days 9 to 23). It was found that there was a 60 to 70% reduction in the number of MALs in the glands that were exposed to HPR. Similarly, a 72% reduction in the number of MALs was observed in glands exposed to 5 n» HPR during the entire culture period. By contrast, the growth and/or maintenance of LA in mammary gland cultures from both Rill and C57BL mice that were exposed to HPR during the developmental phase of LA in lactogenic hormone-containing medium was not impaired, suggesting that HPR may preferentially induce the regression of MALs. Our experiments with organ cultures of MAL-bearing adult mouse mammary glands indicate that these cultures could be an appropriate in vitro system for the study of the direct effects of antineoplastic agents and mechanism(s) of their action on mouse mammary preneoplasia. INTRODUCTION Malignant transformation of the mammary epithelium has been shown to be modified by various exogenous agents such as hormones and vitamins. It has been found that retinoids reduce the incidence of chemical carcinogen-induced mammary tumors of rats (12, 23 to 26). However, while retinyl acetate appeared to enhance the growth of MuMTV3-induced mammary tumors in the GR mouse (43), it did not exhibit any antitumor effect in C3HA"* mice (19). Recently, Dickens and Sorof (8) have reported that the ability of retinoids to prevent chemical carcinogen-induced transformation of mammary glands from non-MuMTV-producing immature BALB/c mice in organ culture is dependent upon the type of carcinogen that is used as the transforming agent. They found that retinylidene dimedone, acting as an antipromotional agent, could prevent mammary gland transformation caused by procarcinogens, but failed to prevent the transformation induced by direct-acting carcinogens or by activated metabolites of procarcinogens (7, 8). Thus, it appears that the antineoplastic prop erties of retinoids, at least with respect to mouse mammary gland cells, may depend upon the strain of mouse (19, 43); the nature of the transforming agent, such as viral (3,27) or chemical (20); and on the hormonal responsiveness of the target tissue (42, 44, 45). In vitro systems capable of prolonged maintenance of isolated mammary glands provide an experimental system wherein the acute and chronic effects of specific agents can be quantitatively examined directly on the mammary epithelium. In view of the importance of MuMTV as an etiological factor in mouse mam mary tumorigenesis, it is important to establish appropriate in vitro systems from select strains of adult mice that vary in the ability to express MuMTV and, thus, differ in their relative risk of developing spontaneous mammary tumors. Tonelli et al. (39) compared the steady-state levels of MuMTV RNA and protein in the mammary gland organ cultures from immature BALB/c, C57BL/6 (MuMTV-negative), and C3H (MuMTV-positive) mice and reported that, although the cultures from all 3 strains had MuMTV-specific RNA, only tissues from C3H mice had detect able levels of gp52, indicating a preferential expression of MuMTV gene expression in the cultures from C3H mice. Other studies (1, 28, 33) have demonstrated that a combination of mammotropic and lactogenic hormones can induce cellular pro liferation and differentiation of mammary epithelium from imma ture mice in organ cultures similar to that seen in intact animals. However, since naturally occurring preneoplastic lesions and overt mammary tumors are observed mostly in adult MuMTVpositive mice, it is important to examine the hormonal respon siveness of naturally occurring MALs4 and of experimentally ' This work was supported by National Cancer Institute Grants CA 25679, CA 17129, and CA 08748. 2To whom requests for reprints should be addressed. Received August 16, 1982; accepted July 6, 1983. 3The abbreviations used are: MuMTV, murine mammary tumor virus; MAL, mammary alveolar lesion; LA, lobuloalveolar structure; HPR, A/-(4-hydroxyphenyl)retinamide; DMSO, dimethyl sulfoxide; IPAH, a combination of insulin, prolactin, aldosterone, and hydrocortisone. 4 In this study, we have defined MALs as distinct alveolar lesions that are