Hyperplastic alveolar nodules of the rat mammary gland: Tumor-producing capability in vivo and in vitro

Cancer Letters,2 (1977)153--160 O'EIseder/North-HollandS6ientificPublishing'Ltd.

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HYPERPLASTICALVEOLAR.NODULESOFTHE R A T MAMMARY G L A N D : T U M O R , P R O D U C I N G CAPABILITY IN ¥ 1 V O AND: IN V I T R O

DILIP SJ[NHA and THOMAS>L. DAO Dep~rtrncn t o f Brea~ Surgery and Breast Cancer Re~,~arch U~dt; Roswell ParI~.Memorial lnstit'u to, N e w Yorl~ State Department o f HeaStl~: Bu;l~falo;New Y ,rk, 14263. (U.S.A.)

(Received30 June 1976) (Revised version received 27 August iJ76)

SUMMARY

Carcinogen-induced hyperplastic alveolar n odules failed to form tumors after transplantation into isologous female ra,ls; instead they developed ductal or ductal-alveolar outgrowths. Mammary glands from mid-pregnant female rats showed similar outgrowths following transplaata~io~ to the isologous hosts. In vitro and in vivo studies failed to demonstl:ate that subcarcinogenic doses of 7,12-dirnethylbenz(a)anthracene Can induce mammary tumorigenesis in the hyperplastic alveolar nodules. Our observations suggest that Carcinogeninduced hyperplastic alveolar nodules are n0t'prene0pl~stic tlesions in mammary carcinogenesis in therat. INTRODUCTION Hyperptastic alveolar nodules (HAN)in the mammary glands of mice and rats can be induced by viruses [ 4,8], chemical carcinogens [ 2,5], and radiation. DeOme and his co-workers transplap ;;ed HAN into the gland-free mammary fat pads of isologous mice and observed tumor formation in many of these trans. plants [4]. This led to the conclusion that hyperplas~ic lesions inthe mouse mammary gland are preneeplastic. Using carcinogen-induced hyperplastic laveolar nodules in the rat, Buying carried out similar transplantation experiments and reported 'tumor' formation in the transplanted hyperpIastic alveolar nodules [t}. This finding, however, ha~ not been confirmed. Studies in our laboratory have not supposed the concept that hyperplas~ic alveolm: nodules are preneoplastic lesions in mammary tumorigenesis in the rat. Thus, we have reported tumor induction in rats without the formation o: hyy..,e~lastic alveolar nodules when 7,12-dimethylbenz(a)anthracene (DMBA) was applied directly to the mammary gland [9], the development of mammary adenocarcinoma in the ducts long before the appearance of hyperplastic alveolar nodules in rats when the carcinogen was given in~ravenously [10],

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and the failure of hyperplastic alveolar nodules to develo;i into tumor~ after transplantation, as described in this paper. These observations led us t,o suggest that; chemically induced mammary carcinogenesis is a directprocess, ilmplying a direct transformation Of normal cells to neoplastic cells and requiring no intermediate step(s). Transplantation of hyperplastic alveolar nodule to mammary gland-.free fat pad naturally requires positive identification of these Iesic,ns in the mammary gland before transplantation. Direct visual identification of hyperplast~k: alveolar nodules in mice is relatively easy since mouse mammary gland is thin and these lesions are characteris~:ically dark brown in colo:~. The hyperplas~ie aiveolar nodules in the rat m~amary gland, however, cannot be v/sualized through the thick layer of fat pad and connective tissues. A simple technique for positive identification of hyperplastic alveolar nodules in situ by direct illumination [3] was developed in this laboratory for tran~plantation studies, We now describe in vitro and in vivo experiments designed to determine t~e tumor-producing capability of DMBA-induced hyperplastic alveolar nodules in the rat mammary gland. MATERIALS A N D M E T H O D S

Female inbred 55-day.old Wistar-Furth rats (A.R. Schmidt, Madison, WJ.sc.) were housed in an air-conditioned roe,m, fed a commercial ration (Rockland diet) and given free access to water. These rats were given a single intravenous injection of an emulsion conta/ning 5 mg DMBA and were used as donors ~or hyperplastic alveolar nodules. These lesions were identified, removed under aseptic conditions, using a technique described previously [3], and used for either in vivo or in vitro studies. In the in vitro studies, the hyperplastic alveolar nodules were cultiva~d in an organ culture system. The technique of organ culture has been re?orted previously [7]. In lhe present study, the ~yperplastie alveolar nodule or norreal mammary glan~ explants were exposed to DMBA in the culture medicare containing hormon,~ supplements (insulin, 0.5 pg/ml; prol~Lctin, 5 #g/mI; estradiol-I7fl, 0.002 ~g/ml; progesterone, 0.1 #g/ml) for either 3 or 6 days.. Control explants were cultured, in the same way except that no carcinogen was added to the medium. All explants were removed after 9 days in culture and were transplanted to the interscapufar fat pad of the i,~ologous t'emale hosts. Each recipient received four explants placed next to each other in the interscapular fat pad. These explants usually coalesce to fc.rm one mass. The rats were killed 90 days after receiving the transplants. At autopsy ~he fotu: pieces of transplant were counted as one unit (Table 1). in the in vivo studies, hyperplas~ic alveolar nodules removed from the donor rats were transplanted immediately to the isologous recipients. ~,acb rat received four hyperplastic alveoIar nodules. For contro:[, pieces of normal mammary gland from the don,,m, which had not received previous carcinogen treatment, were similarly transplanted. Ten days af~.er transplantation, the

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156 recipient rats bearing the transplants were given a single dose of DMBA ~nt~avenously. Three different doses We:regiven to three differen t groups of recipients. The control ~ecipient rats were not given:DMBA~Ninety days after DMBA injection, all rats were killed. Both hos~ marnma~3~ glands and the grafts were removed for whole mount preparations and histologic sections. R E S U L T S A N D DISCUSSION

When hype~plastic alveolar nodule explants were first exposed to DMBA 1 ~g/ml medium for either 3 or 6 days in culture, the explants after transplantation gave rise to tumors in 20 and 213%of the rats bearing surviving grafts, respectively (Table 1). Normal mammary gland exptants treated undex' similar conditions gave rise to tumors ha 57 and 50% of the explants. When a DI~BA dose of less than 1 ~g/ml was used, no mammary tumors developed, either in the hyperplastic alveolar nodule or in the normal mammary gland. If hyperplastic alveolar nodules were more susceptible to the carcinogenic effe~t of DMBA than the normal mammary tissue, one would expect preferential tumorigenesis in hyperplastic alveolar nodules exposed to the subcarcinngenic dose of DMBA. The data from this in vitro experiment do not indicate such a possibility. In the in vivo experiments, the hyperplastic alveolar nodules were transplanted directly to the interscapular fat pads of 55-day-old female WistarFurth isologous recipients. The recipient hosts were either given an emulsion containing no carcinogen (as control.) or an emulsion cot, raining DMBA 10 days after ~eceiving the transplants. Three groups of recipients were each given a different dose of DMBA (0.5 rag/100 g b.w., 1.5 rag/100 g b.w., and 3.0 rag/100 g b.w.). All rats receiving carcinogen doses of either 1.5 or 3.0 rag/100 g b.w. developed tumors in their own mammary glands, but tumors also developed in some hyperplastic alveolar nodule grafts (Table 2). Those hyperplastic ah,eolar nodule expalnts wl~ich did not form tumors developed either ducta] or lobulo-alveolar growth. When the dose of carcinogen was reduced to 0.5 rag/100 g b.w., neither the host gland nor the hyperplasl~ic alveolar nodule transplants developed tumors. This finding illustrates that hyperplastic alveolar nodules do not contain ceil populations which are more susceptible to t]he carcinogen DMBA than are the normal mammary gland cells of the host, The control group in which rats received hyperplastic alveolar nodule transplants but were not given the carcinogen did not develop rumors, either in the host mammary gland or in the transplanted hyperplastic alveolar nodule. Instead, the transplanted hyperplastic alveolar nodules showed ductal growth resembling normal virgin mammary gland (69%) or lobuloalveolar development similar to mammary gland in midpregnant rats (31%) (Figs. 1--3). DeOme, et al. [4] observed in mice that, in most cases, the hyperplastic alveolar nodules following transplantation develop into lobt~loalveolar growth. Such differences in growth patterns in mrs and mice cm~not be readily explained at present.

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Fig. 1. Whole mount preparation of transplanted hyperplastie alveolar nodules, s?,owing Lobulo~alveoiar outgrowth. × 6.5.

Fig. 2. Whole mount preparation of transplanted hyperplastic alveolar nodules, showing mixture of Iobulo-alveolar and ductal outgrowth, x 6.5. These findings clearly indicate that hyperplastic alveolar nodules per se are not precancerous in that they do not give rise to tumors when grafted. Instead, the hyperplastic alveolar transplants develop into ductal outgrowths, resembling mammary glands of virgin rats. This is interesting since a hyperplastic alveolar nodule is composed mainly of alveolar cells. It appears that alveolar cellls have the potential of developing into ductal cells similar to the potential of ducts for alveoli formation. This hypothesis is supported by out observation that

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Fig. 3. Whole mount preparations of tr-~nspl~ntedhyperplastic ~Iveolarnodules, showing ductal outgrowth, comparable to virgin m a m m a r y gland, x 6.5. Dark.areas on upper left and lower leftare originalgrafted hypevplastic alveolar nodules.

Fit~. 4. Whole mount preparation of transplanted mammary glands from a midpregnant rat. Note ductal outgrowth. Dark areas in center are remnants of transplants. × 6,5.

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when pieces of m a m m a r y gland from midpregnant rat were transplanted into isologou~ hosts, they formed ductal outgrowths (Fig. 4 ). It must be mentioned, however, that in all these experiments, we cannotentirely rule out the possibilitythat hyperplastic alveolar n0duleacontain, a mixture Of ductal and alveolar cell populations. The fact that hyperplastic alveolar nodules can transform to neoplasia after being e~,:posedto S e c o n d ~ treatment with carcinogen both in vivo andin vitro suggests that there are Perhaps enough ductal ceils in the hyperplastic alveolar nodule transplant ~;o be transformed into tumors. ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d b y NIH G r a n t C A - 1 4 8 1 2 - 0 3 a n d NIH C o ~ t r a c t N 0 1 - C B . 2 3 8 6 5 f r o m t h e N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s o f Health.

REFERENCES 1 Beuving, L.J. (1965) Mammary tumor formation within outgrowths of transplanted hyperplastie modules from carcinogen-treated rats. J. Natl. Cancer Inst., 40, 1287--t 291. 2 Dao, T.L. (1969) Studies on mechanism of carcinogenesis in the mammary gland. In: Progrea'~ in Experimental Tumor Research, lap. 235--261. Editor: F, Homburger. Karger, Basel, New York. 3 Dao, T.L., Sinha, D., Christakos, S. and Varela, R. {1975) Biochemical characterization of carclnogen-induced mammary hyperplastic alveolar nodule and tumor in the rat. Cancer Res,, 31, 1492--1495. 4 DoOms, K.B., Bern, H.A., Nandi, S,, Pitelka, D. a~d Faullldn, L.J. (i959) Some characteristics of the preneoplastic hyperplast~e alveolar nodules of C~H mice. Cancer Res., 19, 515--520, 5 Faulkin, h.J. (1966) Hyperplastic lesions of mouse mammary gland after treatraent with 3-methylcholanthrene. J. Natl. Cancer Inst., 363, 289~g98. 6 Faulkin, L,J,, Shellabargcr, C.J. and DoOms, K.B. (1967) Hyperplastic lesions of Sprague--Dawley rat mammary glands after x irradiation. J. Natl. Cancer Inst., 39, 449--458. 7 Koyama, H., Sinha, D. and Dao, T.L. (1972) Effects of hormones and 7,12-dimethyl-

benz(a)anthraceneon rat mammary tissuegrown in organ culture.J. Natl.Cancer inst., 48, 1671--1680.

8 Nandi,S. {1963) New method for detectionof mouse mammary tumor virus.I. Influence of foster nursing on incidence of hyperplastic mammalT nodules in BALB/ cCrgl mice. J. Natl. Cancer Inst., 31, 57--73. 9 Sinha, D. and Dao, T.L. (1974) A direct mechanism of mammary ~arcinogenesis induced by 7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst., 53, 841--846. 10 $inha, D. and Dao, T.L. {1975) Site of origin of mammary tumor~ induced by 7,12. dimethylbenz(a)anthracene in the rat. J. Natl. Cancer Inst., 54, 1007~1009.