Rat Models of Premalignant Breast Disease

P1: vendor/GCY/GCZ P2: FMN

Journal of Mammary Gland Biology and Neoplasia (JMGBN) PP030-290990 January 5, 2001 8:9 Style file version Nov. 07, 2000

Journal of Mammary Gland Biology and Neoplasia, Vol. 5, No. 4, 2000

Rat Models of Premalignant Breast Disease

Henry J. Thompson1,3 and Meenakshi Singh2

While a number of agents have been shown to induce mammary carcinogenesis in the rat, pre-malignant stages of the disease have been best characterized in chemically-induced models,specifically those initiated by either 7,12 dimethylbenz[α]anthracene (DMBA)4 or 1-methyl-1-nitrosourea (MNU). In general, it appears that epithelial cells in mammary terminal endbuds or terminal ductules are the targets of carcinogenic initiation, and that a series of mor-phologically identifiable steps are involved in the development of mammary carcinoma. Thepremalignant steps include ductal hyperplasia of the usual type and carcinoma in situ of thecribriform or comedo type; atypical ductal hyperplasia has not been reported. Thus the histo-genesis of lesions occurring in chemically induced mammary carcinogenesis in the rat is similarto that observed in the human; although, the spectrum of lesions observed in the rat is lim-ited. Opportunities to investigate the biological and molecular characteristics of premalignantbreast disease in the rat are presented.

KEY WORDS: Pre-malignancy; breast cancer; experimental model; rat.

INTRODUCTION

The increasing opportunities to target premalig-nant lesions in the prevention of breast cancer, aswell as the importance of premalignant breast lesionsin both risk assessment and in determining patientmanagement options, makes the availability of ani-mal models to study this aspect of disease progressionhighly desirable. Fortunately, animal models for pre-malignant breast disease in the rat already exist, andhave been characterized to various degrees over thelast 40 years. Nonetheless, despite the availability of

1 Center for Nutrition in the Prevention of Disease, AMC CancerResearch Center, Lakewood, Colorado.

2 Department of Pathology, University of Colorado Health Sci-ences Center, Denver, Colorado.

3 To whom correspondence should be addressed to AMC CancerResearch Center, 1600 Pierce Street, Lakewood, Colorado 80214.E-mail: [email protected]

4 Abbreviations: 1-methyl-1-nitrosourea (MNU); 7,12 dimethyl-benz[α] anthracene (DMBA); hyperplastic alveolar nodule(HAN); terminal ductule hyperplasia (TDH); hyperplastic termi-nal end buds (HEB); intraductal proliferation (IDP); terminal endbud (TEB); intraductal proliferation-initiated (IDPi); intraductalproliferation-initiated and promoted (IDPip); ductal carcinomain situ (DCIS); carcinoma in situ (CIS).

such models, relatively little is known about the cellu-lar and molecular events underlying the pathogenesisof premalignant stages of mammary carcinogenesisin the rat. The intent of this paper is to describe an-imal models that are available, to summarize what isknown about the occurrence of premalignant stages ofthe disease in these models, to indicate how prema-lignant disease in the rat compares with the humandisease process, and to identify some of the criticalgaps in knowledge of premalignant breast disease thatcould be investigated in the rat.

HISTOPATHOGENESIS OF BREAST CANCER

The sequential steps most commonly described inthe natural history of breast cancer are: ductal hyper-plasia, atypical ductal hyperplasia, carcinoma in situ,and invasive carcinoma (1,2). Evidence will be pre-sented that the development of mammary carcinomain the rat has a similar natural history (3,4). Thus wewill focus our discussion on premalignant lesions ob-served in the human and the rat, although some con-sideration will also be given to preneoplastic lesionstermed hyperplastic alveolar nodules which are not

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410 Thompson and Singh

Table I. Terminology Used in the Description of Premalignant Breast Lesions in the Rat

Terms used to describe premalignantHuman premalignant lesion General descriptor lesions in the rat

Ductal hyperplasia (DH or IDH) Preneoplasia Terminal ductule hyperplasia (TDH) (5)Premalignancy Intraductal proliferation (IDP) (7)Intraepithelial Hyperplastic end bud (HEB) (34)

neoplasia Ductal and ductal alveolar hyperplasia(DH), (DAH) (36)

Atypical ductal hyperplasia Preneoplasia ADH comparable to the human has not(ADH or AIDH) Premalignancy been reported

Intraepithelialneoplasia

Ductal carcinoma in situ (DCIS) Premalignancy DCIS, cribriform and comedo typesLobular carcinoma in situ (LCIS) Intraepithelial LCIS has not been reported

neoplasia

considered a precursor of mammary carcinomas inthe rat (5–7). It is noteworthy that early, morphologi-cally identifiable pre-cancerous lesions have been re-ferred to in the literature by a variety of names in-cluding dysplasia, preneoplasia, premalignancy, andmore recently mammary intraepithelial neoplasia. Asummary of different terms applied to similar mor-phologically identifiable lesions in the rat is providedin Table I.

MAMMARY CARCINOMA INDUCTIONMODELS IN THE RAT

Chemical-Induced Carcinogenesis. In 1961,Huggins et al. (8) published a method for inducinga high incidence of mammary carcinomas in femalerats using a single dose of carcinogen. Of the threechemical carcinogens on which Huggins reported,7,12 diemethylbenz[α]anthracene (DMBA) wasshown to be the most specific and potent carcinogenfor mammary carcinoma induction. Huggin’s reportushered in what is considered by many investigatorsto be the modern era of research in experimentalmammary carcinogenesis in the rat. The single doseregime reported by Huggins was particularly im-portant since it permitted an operational distinctionbetween the stages of carcinogenic initiation andpromotion/progression. More recently, 1-methyl-1-nitrosourea (MNU) also has been shown to inducespecifically and reproducibly a high incidence ofmammary carcinomas after a single dose giveneither i.v. or i.p.(9,10); this approach was based ona modification of the original model proposed byGullino et al. (11). The biological characteristics of

the DMBA- and the MNU-induced model systemshave been reviewed (12). Common features of bothmodels include reliability of tumor induction, organsite specificity, tumors of ductal histology that are pre-dominantly carcinomas, tumors of varying hormoneresponsiveness, and the potential to examine the pro-cess of tumor initiation and promotion/progression.However, there are also dissimilarities betweenthe two models as summarized by Thompson andAdlakha (10). First, the MNU model provides anexperimental approach for investigating mammarytumorigenesis induced by a direct acting carcinogen;whereas, the DMBA model provides a method forstudying mammary tumorigenesis induced by aproximate carcinogen requiring metabolic activation.Secondly, MNU-induced mammary carcinomasappear more aggressive histologically than theDMBA-induced counterparts although carcinomasinduced by either carcinogen rarely metastasize. Also,the proportion of malignant to benign mammarytumors induced by MNU is higher than for DMBA.MNU-induced mammary carcinomas appear to bemore estrogen dependent; whereas, DMBA-inducedmammary carcinomas appear to be more prolactindependent. In a recent review, the case was arguedthat the merits of the MNU model are such that thereis essentially no good justification for the continueduse of DMBA to induce mammary cancer in rats,unless one is specifically interested in the effectsof carcinogenic hydrocarbons on the mammarygland (13). The reasons cited for this include: thenature of the carcinogenic response, the histologicalcharacteristics of the tumors induced, the simplicityof the tumor induction methodology, and the flexibil-ity of the MNU model in the design of experiments.



Premalignant Breast Disease in the Rat 411

As will be discussed later, in both systems carcinomasof ductal histology that progress from hyperplasiathrough an in situ stage have been reported. Thus,as has been discussed by us before (4), there isconsiderable evidence that both the histology and theintermediary stages of chemically induced mammarycarcinogenesis in the rat are comparable to thoseobserved in the human disease process.

Virally-Induced Carcinogenesis. The injection offemale rats with human adenovirus type 9 (Ad9)induces hyperplasia and tumors in the mammarygland (14,15). However the most common histologi--cal type of tumor observed is fibroadenoma, a be-nign mammary neoplasm. Phyllodes and malignantsarcomas have also been observed (16). It has beenreported that fibroadenomas are derived from mam-mary fibroblasts (collagen type I and vimentin pos-itive cells) and that the sarcomas are derived frommyoepithelial cells (16). Ovariectomy prevents tumordevelopment (17). This model is considered of limitedinterest for the investigation of human premalignantbreast disease, and we will not discuss it further.

Oncogene-Induced Carcinogenesis. In a rel-atively recent method development, Gould andcoworkers have devised an experimental approach toincorporate selected genes in situ into mammary ep-ithelial cells(18,19). The gene of interest is subclonedinto a retroviral vector, which after further manipu-lation, is infused into the central ducts of all 12 mam-mary glands of the rat. After infusion, the viral parti-cles enter ductal mammary cells that abut the lumen.Following reverse transcription, virally encoded DNAis incorporated into the mammary genome. Using thisapproach, these investigators have reported that theactivated ras or mutated neu oncogenes induce mam-mary carcinomas. In the case of the neu oncogene,premalignant lesions, specifically in situ carcinomaswith a cribiform-comedo morphology, were also de-tected(19). This approach could offer significant op-portunities for investigating the pathogenetic basis ofpremalignant breast disease.

Radiation-Induced Mammary Carcinogenesis.The rat mammary gland is exquisitely sensitive to radi-ation induced carcinogenesis. Radiation can be eitherlocally delivered or provided as whole body radia-tion (20–22). It appears that hyperplastic lesions areinduced, and they are reported to be similar to hyper-plastic alveolar nodules observed in mice (23). How-ever, the characterization of the natural history of thedisease process induced by radiation is very limited.Because of the limited characterization of premalig-nant lesions in this system, we will not be discussing

it further. If these lesions are characterized in futurestudies, radiation-induced models could be a fertilearea for investigation since radiation exposure is aknown etiologic factor in the human disease (24,25).

Based on this brief overview of experimental ap-proaches to inducing mammary carcinogenesis in therat, the only models in which there has been substan-tive investigation of the natural history of the diseaseis in chemically-induced models. The remainder of thispaper will be limited to mammary carcinogenesis in-duced by either DMBA or MNU.

PREMALIGNANT STAGESOF CHEMICALLY-INDUCEDMAMMARY CARCINOGENESIS

DMBA-Induced Mammary Carcinogenesis. Fol-lowing the publication of the method of Hugginset al. (8) for the induction of mammary carcinomas byDMBA, a number of laboratories sought to identifythe origins of the lesions induced and the histopatho-genesis of the disease process. For a number of yearsthere were questions about whether a precursor le-sion existed in this model system, and about the ma-lignant potential of the precursor lesion(s). Some ofthese questions are still unanswered.

In 1965, Middleton proposed that carcinomasinduced by DMBA originated in epithelial cells ofmammary ducts, ductules and end-buds; this obser-vation was deduced via examination of the histologicpattern of lesions found adjacent to palpable tumors(26). However, inspection of mammary gland whole-mounts from DMBA-treated rats also revealed a highfrequency of hyperplastic alveolar nodules (HAN)which led Beuving and coworkers to hypothesize thatHAN, which had been shown to be precursors ofmammary carcinomas in virally-induced mouse mod-els (27), were precursor lesions in the DMBA ratmodel as well (28). Subsequently Beuving reportedthat HAN obtained from DMBA-treated rats, whentransplanted into compatible recipient animals, gaverise to mammary carcinomas (29). This finding sup-ported a precursor role for HAN in the genesis ofmammary carcinoma, although the HAN as well asthe HAN outgrowths that arose from transplantationdid not require ovarian hormones for their mainte-nance. The majority of DMBA-induced mammarycarcinomas are hormone dependent. Subsequently,other workers failed to confirm Beuving’s finding thatHAN gave rise to mammary carcinoma (30). In an in-teresting series of papers, Dao and colleagues showed




(a) that mammary carcinomas were of ductal origin;(b) that their appearance preceded the appearanceof HAN in rats intravenously injected with DMBA;(c) that local application of DMBA to the mammarygland resulted in the induction of mammary carcino-mas in the absence of HAN; and (d) that HAN trans-planted into animals that were subsequently treatedwith carcinogen were no more susceptible to carcino-genic transformation than transplanted ducts fromsimilarly treated animals (6,31,32). These studies ledDao and colleagues to conclude that HAN were nota precursor lesion for DMBA-induced mammary car-cinomas, and that malignant changes as a result ofcarcinogen treatment led directly to tumor forma-tion by progressive growth of the transformed cells,without any intervening lesions preceding the appear-ance of carcinomas (32). During this same time frame,Haslam et al. (33) reported that HAN from DMBAtreated animals failed to develop into mammary car-cinoma at a frequency different than that of randomlytransplanted ducts from the same animals; these au-thors also reported that another type of dysplasia, de-scribed as a terminal ductule hyperplasia (TDH), wasobserved in DMBA treated rats. TDH preceded theappearance of mammary carcinomas, and while thenumber of TDH investigated was small, 50% of trans-planted TDH gave rise to palpable mammary tumorsthat subsequently regressed (33). In addition, in largerTDH, a gradation from focal epithelial hyperplasiato carcinoma in situ was observed. These findingswere consistent with those of Middleton and Dao, al-though they suggest that an intermediate hyperplas-tic step exists in the natural history of the disease,and that these hyperplasias have variable potential tobecome carcinomas. During this period, Russo et al.also provided a comprehensive account of the patho-genesis of DMBA-induced mammary carcinomas thatdescribed their origin as a multifocal phenomenonin intraductal proliferations (IDP) arising in termi-nal end bud epithelium (TEB) (7). TEB are terminalductal structures ending in bulbous clubs that gener-ally undergo cleavage and develop into alveoli. Russoalso hypothesized that HAN originated from alveo-lar buds and did not give rise to mammary carcino-mas. In a later study Purnell reported the inductionof HAN and hyperplastic terminal end buds(HEB)by DMBA treatment. HEB were detectable within7 days of carcinogen treatment and preceded the ap-pearance of mammary carcinomas; whereas, HAN de-veloped relatively late and became more numerouswith the passage of time (34). This study implicated

terminal end bud hyperplasia in the pathogenesis ofDMBA-induced mammary carcinomas.

In describing the histopathogenesis of DMBA-induced mammary carcinomas, Russo et al. quantifiedthe frequency of occurrence of IDP over time follow-ing DMBA administration (7). IDP were reported tobe distinguishable from TEB by their size, IDP beingmore than twice as large as TEB, and by their ho-mogenous cell composition which consisted predom-inately of intermediate cells. The number of IDP wassuggested to be approximately 200 per animal; giventhat these animals develop only 5–6 carcinomas, theexistence of two types of IDP referred to as initiatedIDP (IDPi) and initiated and promoted IDP (IDPip)was hypothesized (35). IDPi were reported not toprogress to carcinomas and to have histologic featuresthat distinguish them from IDPip which do progressto carcinomas. Specifically, IDPi failed to elicit a stro-mal reaction; whereas, IDPip elicited a marked stro-mal reaction consisting of collagen deposition and in-filtration by mast cells and lymphocytes (35). IDPip

were reported to progress to carcinoma in situ andinvasive cancer. This hypothesis differs from that ofDao in which direct carcinogenic transformation with-out an intermediate step was proposed. Further test-ing of the hypothesis advanced by Russo et al. couldlead to new opportunities to identify the genetic andepigenetic factors that regulate premalignant diseaseprogression; however, a vigorous test of fundamentalaspects of this hypothesis using transplantation tech-niques has not yet been reported.

In summary, several types of finding support theconcept that, in the DMBA model, epithelial cells interminal end buds or terminal ductulolobular unitsare the targets of carcinogenic initiation, and thata series of morphologically identifiable steps are in-volved in the development of mammary carcinoma.These steps involve ductal hyperplasia, carcinomain situ (cribiform and papillary types) and invasivecarcinoma (cribiform and papillary as the predomi-nant types). That there are populations of carcinogeninitiated hyperplasias that have different potentialsfor promotion and progression to cancer is suggestedby the literature, but requires further investigation.Chemically-induced HAN do not appear to representpremalignant lesions in the rat.

MNU-Induced Mammary Carcinogenesis. In theoriginal report of the MNU model by Gullino (11), alimited amount of evidence was presented indicatingthat the mammary carcinomas were of ductal origin,a finding that paralleled Middleton’s report on the




origin of mammary carcinomas induced by DMBA(26). Additional studies of the origin and histopatho-genesis of the MNU-induced disease process werenot reported for approximately 15 years. In 1991,Anderson and coworkers reported the occurrence ofmicroscopically identifiable dysplasia in TEB within1 week following MNU administration (36). The find-ing that dysplasia arose in TEB paralleled the ob-servation of Russo et al. (7) in the DMBA model.Anderson et al. also reported the occurrence of duc-tal and ductal alveolar hyperplasia and HAN between3 and 6 weeks post carcinogen with adenocarcinomaarising from the proximal and distal ductal network.In the same year Sakai and Ogawa reported the occur-rence of IDP as a very early change in the carcinogenicprocess(37). In 1992, Crist et al. (38) determined thefrequency of occurrence of ductal carcinoma in situin animals injected with MNU. Between 22 and 45days following 2 i.v. injections of MNU spaced oneweek apart, an 87% incidence of ductal carcinoma insitu (DCIS) was observed in the absence of invasivecancer. Thus, based on a compilation of the results ofseveral reports, the histogenesis of lesions occurringin the MNU model appears similar to that observedin the human.

SIMILARITIES AND DIFFERENCES IN THEDMBA AND THE MNU MODELS

There appear to be many similarities in thehistopathogenesis of mammary carcinomas inducedby administration of either DMBA or MNU despitethe confusion that is caused by the use of differentterminology to describe what appears to be the samelesion. Thus as summarized in Table I, lesions are ob-served in both models that appear to correspond toductal hyperplasia of the usual type in humans. Thesehyperplastic ductal lesions have been termed termi-nal ductule hyperplasia, hyperplastic end buds, ductaland ductal alveolar hyperplasia, and intraductal pro-liferations. In the studies we reviewed, there has beenno discussion of whether the atypical ductal hyper-plasia that occurs in the human is observed in the rat.Not all investigators have reported the occurrence ofcarcinoma in situ (CIS), and when CIS has been re-ported, the types of CIS found have not always beendescribed, although the dominant forms appear to becribriform and comedo types.

There are three areas in which the reports of vari-ous investigators do not agree, for reasons that may bedue to differences in experimental approach among

laboratories rather than actual differences in the dis-ease process in these model systems. The three areasare: the location at which tumors arise in the mam-mary gland tree, the actual number of premalignantlesions that occur per animal, and malignant poten-tial of the induced ductal hyperplasia. Briefly, it ap-pears that hyperplasia and microtumors have been re-ported to occur primarily in the periphery of the glandin the DMBA model; whereas, they are reported tobe disseminated throughout the gland in the MNUmodel. This finding could be important if it indicatesthat mammary carcinomas arise not only within TEB,but also from the more differentiated structures thatare derived from TEB. There also is considerable vari-ability in the number of premalignant lesions reportedper rat in various studies. This variability may be dueto the type and/or size of the lesions that various in-vestigators have included in such counts rather than todifferences in the disease process induced by DMBAor MNU. The issue of quantification of lesion occur-rence is not a trivial point since there are several or-ders of magnitude variation in the numbers of lesionsper animal reported by different investigators. Thesedifferences impact the case made for or against theoccurrence of populations of ductal hyperplasia withdiffering potentials to upgrade to carcinoma. Relatedto this problem is the report in the DMBA model thathyperplasia with different potentials to develop intocarcinoma can be classified separately histologically;a similar observation has not been reported in theMNU model.

AN ALTERNATIVE MODEL FOR STUDYINGPREMALIGNANT BREAST DISEASEIN THE RAT

In 1995, our laboratory reported the develop-ment of a mammary carcinogenesis model in whichthere was a rapid induction of premalignant and ma-lignant mammary gland lesions (39) achieved by in-jecting rats with the carcinogen MNU at 21 insteadof 50 days of age. We reasoned that this age wouldbe optimal for carcinogenic initiation because of thelarge number of TEB, which have been reported to bethe target of carcinogenic initiation, that are presentin the mammary gland at this age, and that events nec-essary for carcinogenic initiation, promotion, and pro-gression would be accelerated immediately followingcarcinogen treatment due to the rapid growth and de-velopment of the mammary gland between 21 and55 days of age. Mammary intraductal proliferations




measuring greater than 1 mm2 were identifiablewithin 14 days of carcinogen administration. Whilethis timeframe of occurrence is in agreement withsome reports (7,37), it is clear that these intraductalproliferations arose earlier than 14 days post carcino-gen since Russo and coworkers (40) have reported theability to discriminate between TEB and IDP whenIDP were approximately 0.2 mm2 in diameter, andPurnell (34) and Anderson et al. (36) have reporteddetecting abnormalities in TEB as early as 7 days postcarcinogen treatment. IDP were detected in the ab-sence of either carcinoma in situ or microcarcinoma.However, by 21 days post carcinogen both DCIS andcarcinoma also were observed. By 35 days post car-cinogen greater than 90% of animals injected with 50mg MNU/ kg body weight had a spectrum of premalig-nant and malignant lesions greater than 1 mm2 in theirabdominal-inguinal mammary gland chains. Many ofthe carcinomas observed had a prominent CIS com-ponent, which is consistent with the hypothesis thathyperplasia progress to carcinoma via a CIS interme-diary step. Thus, the temporal sequence of events in-volved in the emergence of invasive carcinomas gen-erally appears to progress from ductal hyperplasia toductal carcinoma in situ to invasive carcinoma as hasbeen reported in the DMBA model. Nonetheless, asdiscussed in (41), it is possible that some hyperplasiasbecome invasive carcinomas rapidly and without dis-playing all the histological features of CIS. The advan-tages of this model include 1) the ability to identifyneoplasia visually, and to excise these lesions eitherin situ for transplantation or molecular studies or fromwholemount preparations for subsequent molecularand histochemical analyses as well as for histologicdiagnosis, 2) the high prevalence of these lesions ina group of animals after a short latency; and 3) thelocal invasion of adjacent lymph nodes and muscle bysome of the microcarcinoma.

Because of the importance of understanding howthe pathogenesis of the disease process in the rat re-lates to that occurring in the human, the lesions fromthis rat model were recently described using the ter-minology and criteria applied to human breast dis-eases (4). It was found that the hyperplastic lesionsobserved in the rat are best classified as hyperpla-sia of the usual type and in general are compara-ble to the types of hyperplasia observed in the hu-man breast. In this model, hyperplasia ranged the fullgamut from mild to florid and varying grades of hy-perplasia could be observed in a single field. How-ever, atypical ductal hyperplasia comparable to thatobserved in humans was not observed. CIS was also

observed in the rat model, a finding consistent withother reports (3,38). While the histologic spectrumof CIS observed in the rat was limited compared tothat seen in humans, the lesion types observed in ratsrepresent commonly observed types in humans. Crib-riform CIS was most common followed by CIS with acomedo pattern; however, the nuclear grade of theselesions was low. Thus, they are not truly comparableto the comedo CIS that occurs in the human breast.Although pure micropapillary CIS was not seen in therat, there was some attempt at luminal micropapillaeformation in ducts that showed cribriform CIS. Withinthe 35 day timeframe following carcinogen adminis-tration neither lobular hyperplasia nor lobular car-cinoma in situ were observed. Thus while there aresimilarities in the histopathogenesis of this short la-tency model to the human, there are differences aswell. They include the limited spectrum of lesions thatare observed in the rat and the low nuclear grade oflesions in the rat relative to nuclear grade observedin humans. We speculate that the period of observa-tion may account, at least in part, for these differencesand that increasing the dose of carcinogen used to in-duce the disease process would result in lesions witha higher nuclear grade.

APPROACHES TO STUDYINGPREMALIGNANT LESIONS IN THE RAT

In developing the alternative model discussedearlier, we have devised approaches to manipulatingthe mammary gland that facilitate the investigationof premalignant lesions (39,42). We judge that theseapproaches can be applied to rats of all ages irrespec-tive of the carcinogenic insult to which they have beenexposed. We briefly summarize these approaches andillustrate them using tissue specimens from the shortlatency model (43). Problems are encountered in vi-sualizing the occurrence of premalignant lesions inthe cervical-thoracic mammary gland chain due to thepresence of muscle that lies between the second andthird gland. Therefore, we recommend focusing stud-ies on the abdominal-inguinal mammary gland chain.While there are many reports in which the mammaryglands have been excised from the skin, after bothhave been removed from the animal, we recommendthe excision of the gland in situ as detailed in refer-ence (39). The excised gland can be spread on a glassslide to visualize glands 4–6 (Fig. 1). If the gland is tobe used for excising lesions that will subsequently betransplanted, the entire procedure can be done using




Fig. 1. (A). An abdominal-inguinal mammary gland chain as it is being excised from the animal. Bar, 1 mm. (B). An in situ preparationof an abdominal-inguinal mammary gland chain spread out on a glass slide and subjected to a bright light source from beneath thepreparation. The fifth mammary gland was cannulated via the nipple and infused with a solution of methylene blue. Note the ability tovisualize the mammary gland structures in both the unstained (arrow) and dye-infused areas of the wholemount preparation.




aseptic technique. Premalignant lesions can be identi-fied either by illuminating the tissue using principles ofdark field microscopy, or via the injection of dye intothe glands to be inspected (29) (Fig. 1B) (44). The le-sions excised also can be used for molecular analysesusing appropriate microtechniques. If assays appliedto the glands are to be histological or immunohis-tochemical, the excised gland, mounted on the glassslide, can be submerged in fixative for 18–24 hours.We routinely fix both abdominal-inguinal mammarygland chains from each rat. One chain is fixed in 10%neutral buffered formalin; whereas, the other chainis fixed in methacarn. The glands are subsequentlycleared and stained using alum carmine. The glandsare then put into bags containing methyl salicylate andsealed. These specimens can then be photographeddigitally. The quality of the specimens obtained is il-lustrated in Fig. 2A. These wholemounts can be in-spected, and various premalignant lesions identified(Fig. 2A and 2B). The wholemounts can also be eval-uated digitally and various morphometric parametersestimated, for example, percent of the fat pad occu-pied by epithelium, ductal extension, and mammarygland mass. Lesions identified in wholemounts alsocan be excised and further processed for histolog-ical, immunohistochemical, and molecular analyses.The histology of lesions identified in Fig. 2A is shownin Fig. 2C. We have assessed proliferation, apopto-sis, estrogen and progesterone receptor status, vari-ous cell cycle regulatory proteins, and angiogenesis-related proteins in samples obtained in this manner.We have also used specimens obtained in this way forDNA extraction and RFLP analyses. Thus, by pro-cessing the mammary gland in the manner described,it is possible to investigate the biological and molec-ular characteristics of premalignant lesions in the rat.

BIOLOGIC CHARACTERISTICSOF CHEMICALLY-INDUCEDPREMALIGNANT LESIONS

Little is known about the biologic characteristicsof chemically-induced mammary gland premalignantlesions in the rat. From work using the DMBA model,it is known that a proportion of transplanted ductalhyperplasias can develop into palpable mammary tu-mors, but because these tumors regressed, the ovarianhormone dependence of the resulting tumors couldnot be assessed (33). In the MNU model, to our knowl-edge, there have been no transplantation studies ofpremalignant lesions. However, the effects of ovariec-tomy on the prevalence of premalignant and malig-

nant lesions has been reported in the short latencymodel described earlier (45). Those data indicate thatpremalignant lesions induced by MNU can be bothdependent on [65–80% depending on criteria used(43)] and independent of ovarian steroids. This findingimplies that autonomy from hormonal control may ei-ther be a preexisting characteristic of some mammaryepithelial cells transformed by carcinogenic initiation,or that this property is conferred to some mammaryepithelial cells at the time of carcinogenic initiation.However, further studies are needed to more fully ex-plore these possibilities.

A major opportunity that exists in this area isto further characterize the biological behavior of pre-malignant lesions induced in the rat using transplanta-tion technology. The use of transplantation techniquesin rat models has been very limited, despite the po-tential of this approach to render new insights aboutpremalignancy. A few of the questions that wouldbe intriguing to address include, 1) can stable duc-tal hyperplastic outgrowths be established from pre-malignant lesions in the rat ?, 2) do populations ofIDP, identifiable by histological criteria, differ in theirprobability of progressing to carcinoma ?, 3) what ge-netic and/or epigenetic factors account for differencesin this potential if the concept is validated ?, and 4) doIDP that emerge in ovariectomized animals progressto mammary carcinoma, and are the carcinomas au-tonomous of ovarian steroids for their growth andmaintenance?

It is important to note that recent activities in thescientific community have been intended to promotethe use of transplant technology to study mammarygland biology and neoplasia. These efforts include thepublication of a techniques manual which includes achapter describing transplantation methodology (46),and the production of a video illustrating this tech-nique (http://www.biology.ucsc.edu/mamvid). We alsonote that visualization of premalignant lesions in situis essential for lesion transplantation; several strate-gies have been proposed for this purpose and are de-scribed in the following references (29,44).

CELLULAR AND MOLECULARCHARACTERISTICS OFPREMALIGNANT LESIONS

There is a dearth of knowledge about the cellularand molecular characteristics of chemically-inducedpremalignant or malignant mammary glands lesionsin the rat. This topic has been reviewed recently (47).What is known can be summarized as follows: It




Fig. 2. (A). A fixed and stained mammary gland wholemount preparation in which a number of premalignant and malignant mammarygland lesions can be seen. At this magnification some regions of the wholemount are out of focus; none-the-less, note the ability to easilyidentify potentially abnormal hyperplastic foci. Bar, 10 µM. (B). The subgross morphology of lesions identified in Panel A. It is not possibleto accurately predict the diagnosis of a lesion based on its subgross morphology. (C). The histology of the lesions shown in Panel B. Bars,10 µM.




appears that chemically-induced mammary carcino-mas, like their human counterparts, have altered ex-pression of TGFα, ErbB2, cyclin D1 and gelsolin, butdo not have misregulated p53 activity as is commonlyseen in the human disease (48,49). However, therehave been no reports of whether these alterationsare observed in premalignant stages of the diseaseprocess. A G to A transition mutation in codon 12of the Ha-ras gene is frequently observed in MNU-induced mammary carcinomas (50). Of particular in-terest is the report that 60 to 80% of IDP’s observedin MNU treated rats also have this mutation (37,51).Given that these investigators reported that similarproportions of the mammary carcinomas that wereinduced had the same Ha-ras mutation, the observa-tions are consistent with the hypothesis that IDP is infact a premalignant lesion in this model system. It isalso of interest that Archer and coworkers reportedthat host factors can affect whether IDP progress toductal carcinoma in situ or disappear; this observationimplies that IDP are not necessarily fully transformedcarcinoma cells as suggested by Dao; rather, IDP maybe comprised of populations of cells with varying po-tentials to become malignant as is observed in themouse. In the DMBA model, approximately 16% ofmammary adenocarcinomas are observed to have amutation (CAA to CTA) in codon 61 of Ha-ras; nostudies of the frequency of this mutation in prema-lignant mammary lesions have been reported. How-ever, such mutations are uncommon in human breastcancer (24).

It is clear that additional work is required to clar-ify the pathogenetic basis of the disease process in-duced in the rat mammary gland by chemical carcino-gens. A combination of immunohistochemical and/ormolecular techniques can be applied to excised lesionsor lesion outgrowths if transplantation strategies areused. Determining the role of alterations in cell cycleregulation in the histopathogenesis of the disease is agoal that is feasible to pursue given the availability ofappropriate animal models and molecular reagents.In addition, due to the importance of neovasculariza-tion to progression of premalignant lesions to carcino-mas with palpable dimensions, investigating the roleof angiogenesis in premalignant and malignant stagesof this disease process also has considerable merit.

CONCLUSIONS

While rat models for the investigation of prema-lignant stages of breast disease have existed for over

40 years, they have not been widely studied; chem-ically induced models are currently the best char-acterized. In these models an opportunity exists toapply currently available cellular and molecular tech-niques to gain new insights about this aspect of breastcarcinogenesis. Of the many important issues thatshould be addressed, a number of critical questionsmerit particular attention. They include: 1) do all duc-tal hyperplasias have similar potential to progress tomalignancy? If hyperplasias do vary in this regard,as the results reviewed imply, then it will be impor-tant to identify the genetic and/or epigenetic factorsthat account for these differences, and to determinewhether there are histological characteristics that canbe used to identify lesions with differing potentials toprogress to invasive cancers; 2) at what stage of his-tologic progression is angiogenesis induced, is the an-giogenic response during premalignant breast diseasegreater than that observed during mammary glanddevelopment, pregnancy or lactation, and is there anassociation between the angiogenic potential of a pre-malignant breast lesion and its ability to progress tomalignancy ?; and 3) at what stage of histologic pro-gression is the balance between cell proliferation andapoptosis altered, and what changes in the regulationof the cell cycle and apoptosis underlie the observedderegulation of these cellular processes? By investi-gating these problems, it is likely that new approachesto the prevention of premalignant breast disease willbe identified.

ACKNOWLEDGMENTS

This work was supported in part by PHS grantsCA52626 and CA69241 from the National Cancer In-stitute. We wish to thank John McGinley for his helpin preparing the text figures.

REFERENCES

1. S. R. Wellings, H. M. Jensen, and R. G. Marcum (1975). Anatlas of subgross pathology of the human breast with specialreference to possible precancerous lesions. J. Natl. Cancer Inst.55:231–273.

2. S. R. Wellings (1980). Development of human breast cancer.Adv. Cancer Res. 31:287–314.

3. J. Russo, B. A. Gusterson, A. E. Rogers, I. H. Russo, S. R.Wellings, and M. J. van Zwieten (1990). Comparative study ofhuman and rat mammary tumorigenesis. Lab. Invest. 62:244–278.




4. M. Singh, J. N. McGinley, and H. J. Thompson (2000). A com-parison of the histopathology of premalignant and malignantmammary gland lesions induced in sexually immature rats withthose occurring in the human. Lab. Invest. 80:221–231.

5. S. Z. Haslam (1980). The effect of age on the histopathogene-sis of 7,12-dimethylbenz(α)-anthracene-induced mammary tu-mors in the Lewis rat. Int. J. Cancer 26:349–356.

6. D. Sinha and T. L. Dao (1975). Site of origin of mammary tu-mors induced by 7,12-dimethylbenz(α)anthracene in the rat.J. Natl. Cancer Inst. 54:1007–1009.

7. J. Russo, J. Saby, M. Isenberg, and I. H. Russo (1977). Patho-genesis of mammary carcinomas induced in rats by 7,12-dimethylbenz[α]antrhacene. J. Natl. Cancer Inst. 59:435–445.

8. C. B. Huggins, L. C. Grand, and F. P. Brillantes (1961). Mam-mary cancer induced by a single feeding of polynuclear hydro-carbons and its suppression. Nature (London), 189:204–207.

9. D. L. McCormick, C. B. Adamowski, A. Fiks, and R. C. Moon(1981). Lifetime dose-response relationships for mammarytumor induction by a single administration of N-methyl-N-nitrosourea. Cancer Res. 41:1690–1694.

10. H. J. Thompson and H. Adlakha (1991). Dose-responsive in-duction of mammary gland carcinomas by the intraperitonealinjection of 1-methyl-1-nitrosourea. Cancer Res. 51:3411–3415.

11. P. M. Gullino, H. M. Pettigrew, and F. H. Grantham (1975).N-nitrosomethylurea as mammary gland carcinogen in rats.J. Natl. Cancer Inst. 54:401–414.

12. C. W. Welsch (1985). Host factors affecting the growth ofcarcinogen-induced rat mammary carcinomas: A review andtribute to Charles Brenton Huggins. Cancer Res. 45:3415–3443.

13. H. J. Thompson and M. B. Sporn (2000). Mammary cancer inrats. In B. A. Teicher (ed.), Tumor Models in Cancer Research,Churchill Livingstone, New York (in press).

14. N. Jonsson and J. Ankerst (1977). Studies on adenovirus type9-induced mammary fibroadenomas in rats and their malignanttransformation. Cancer 39:2513–2519.

15. J. Ankerst and N. Jonsson (1989). Adenovirus type 9-inducedtumorigenesis in the rat mammary gland related to sex hor-monal state. J. Natl. Cancer Inst. 81:294–298.

16. R. Javier, K. Raska, Jr. , G. J. MacDonald, and T. Shenk(1991). Human adenovirus type 9-induced rat mammary tu-mors. J. Virol. 65:3192–3202.

17. R. Javier and T. Shenk (1996). Mammary tumors induced byhuman adenovirus type 9: A role for the viral early region 4gene. Breast Cancer Res. Treat. 39:57–67.

18. B. Wang, W. S. Kennan, J. Yasukawa-Barnes, M. J. Lindstrom,and M. N. Gould (1991). Frequent induction of mammary car-cinomas following neu oncogene transfer into in situ mammaryepithelial cells of susceptible and resistant rat strains. CancerRes. 51:5649–5654.

19. M. N. Gould (1993). Cellular and molecular aspects of the mul-tistage progression of mammary carcinogenesis in humans andrats. Semin. Cancer Biol. 4:161–169.

20. J. J. Broerse, L. A. Hennen, W. M. Klapwijk, and H. A. Solleveld(1987). Mammary carcinogenesis in different rat strains afterirradiation and hormone administration. Int. J. Radiat. Biol.Related Stud. Phys. Chem. Med. 51:1091–1100.

21. D. W. van Bekkum and J. J. Broerse (1991). Induction of mam-mary tumors by ionizing radiation. Radiat. Environ. Biophys.30:217–220.

22. D. A. Kantorowitz, H. J. Thompson, and P. Furmanski (1995).Effect of high-dose, fractionated local irradiation on MNU-induced carcinogenesis in the rat mammary gland. Carcino-genesis 16:649–653.

23. L. J. Faulkin, C. J. Shellabarger, and K. B. DeOme (1967). Hy-perplastic lesions of Sprague-Dawley rat mammary glands afterX irradiation. J. Natl. Cancer Inst. 39:449–458.

24. C. E. Land (1997). Radiation and breast cancer risk. Prog. Clin.Biol. Res. 396:115–124.

25. M. Tokunaga, C. E. Land, S. Tokuoka, I. Nishimori, M. Soda,and S. Akiba (1994). Incidence of female breast cancer amongatomic bomb survivors, 1950–1985. Radiat. Res. 138:209–223.

26. P. J. Middleton (1965). The histogenesis of mammary tumors in-duced in the rat by chemical carcinogens. Brit. J. Cancer 19:830–839.

27. D. Medina (1996). Preneoplasia in mammary tumorigenesis.Cancer Treat. Res. 83:37–69.

28. L. J. Beuving, L. J. Faulkin, Jr. , K. B. DeOme, and V. V. Bergs(1967). Hyperplastic lesions in the mammary glands of Sprague-Dawley rats after 7,12-dimethylbenz[α]anthracene treatment.J. Natl. Cancer Inst. 39:423–429.

29. L. J. Beuving (1968). Mammary tumor formation within out-growths of transplanted hyperplastic nodules from carcinogen-treated rats. J. Natl. Cancer Inst. 40:1287–1291.

30. L. J. Beuving (1969). Effects of ovariectomy on preneoplasticnodule formation and maintenance in the mammary glands ofcarcinogen-treated rats. J. Natl. Cancer Inst. 43:1181–1189.

31. D. Sinha and T. L. Dao (1974). A direct mechanism of mammarycarcinogenesis induced by 7,12-dimethyl-benz(α)anthracene.J. Natl. Cancer Inst. 53:841–846.

32. D. Sinha and T. L. Dao (1977). Hyperplastic alveolar nodulesof the rat mammary gland: Tumor-producing capability in vivoand in vitro. Cancer Lett. 2:153–160.

33. S. Z. Haslam and H. A. Bern (1977). Histopathogenesisof 7,12-diemthylbenz(α)anthracene-induced rat mammary tu-mors. Proc. Natl. Acad. Sci. U.S.A. 74:4020–4024.

34. D. M. Purnell (1980). The relationship of terminal duct hy-perplasia to mammary carcinoma in 7,12-dimethylbenz(α)anthracene-treated LEW/Mai rats. Amer J. Pathol. 98:311–324.

35. J. Russo and I. H. Russo (1991). Boundaries in mammary car-cinogenesis. Basic Life Sci. 57:43–57.

36. C. H. Anderson, R. A. Hussain, M. C. Han, and C. W.Beattie (1991). Estrous cycle dependence of nitrosomethylurea(NMU)-induced preneoplastic lesions in rat mammary gland.Cancer Lett. 56:77–84.

37. H. Sakai and K. Ogawa (1991). Mutational activationof c-Ha-ras genes in intraductal proliferation induced byN-nitroso-N-methylurea in rat mammary glands. Int. J. Cancer49:140–144.

38. K. A. Crist, B. Chaudhuri, S. Shivaram, and P. K. Chaudhuri(1992). Ductal carcinoma in situ in rat mammary gland. J. Surg.Res. 52:205–208.

39. H. J. Thompson, J. N. McGinley, K. Rothhammer, and M. Singh(1995). Rapid induction of mammary intraductal proliferations,ductal carcinoma in situ and carcinomas by the injection of sex-ually immature female rats with 1-methyl-1-nitrosourea. Car-cinogenesis 16:2407–2411.

40. I. H. Russo and J. Russo (1978). Developmental stage of therat mammary gland as determinant of its susceptibility to7,12-dimethylbenz[α]anthracene. J. Natl. Cancer Inst. 61:1439–1449.

41. H. J. Thompson, J. N. McGinley, P. Wolfe, M. Singh, V. E. Steele,and G. J. Kelloff (1998). Temporal sequence of mammary intra-ductal proliferations, ductal carcinomas in situ and adenocarci-nomas induced by 1-methyl-1-nitrosourea in rats. Carcinogen-esis 19:2181–2185.




42. J. N. McGinley, K. K. Knott, and H. J. Thompson (2000). Effectof fixation and epitope retrieval on BrdU indices in mammarycarcinomas. J. Histochem. Cytochem. 48:355–362.

43. H. J. Thompson, J. McGinley, K. Rothhammer, and M. Singh(1998). Ovarian hormone dependence of pre-malignant andmalignant mammary gland lesions induced in pre-pubertal ratsby 1-methyl-1-nitrosourea. Carcinogenesis 19:383–386.

44. T. L. Dao, S. S. Chistakos, and R. Varela (1975). Biochemicalcharacterization of carcinogen-induced mammary hyperplasticaveolar nodule and tumor in the rat. Cancer Res. 35:1128–1134.

45. W. Z. Wei, R. Pauley, D. Lichlyter, H. Soule, W. P. Shi, G. Calaf,J. Russo, and R. F. Jones (1998). Neoplastic progression ofbreast epithelial cells—a molecular analysis. Brit. J. Cancer78:198–204.

46. L. J. Young (2000). The cleared mammary fat pad and thetransplantation of mammary gland morphological structuresand cells. In M. Ip and B. Asch (eds.), Methods in MammaryGland Biology and Breast Cancer Research, Plenum MedicalBook Company, New York (in press).

47. D. Medina and H. J. Thompson (2000). A comparison of thesalient features of mouse, rat and human mammary tumorige-nesis. In M. Ip and B. Asch (eds.), Methods in Mammary GlandBiology and Breast Cancer Research, Plenum Medical BookCompany, New York (in press).

48. K. Ogawa, Y. Tokusashi, and I. Fukuda, (1996). Absence of p53mutations in methylnitrosourea-induced mammary tumors inrats. Cancer Detect. Prev. 20:214–217.

49. K. Kito, T. Kihana, A. Sugita, S. Murao, S. Akehi, M. Sato, M.Tachibana, S. Kimura, and N. Ueda (1996). Incidence of p53and Ha-ras gene mutations in chemically induced rat mammarycarcinomas. Mol. Carcinogensis 17:78–83.

50. S. Sukumar, V. Notario, D. Martin-Zanca, and M. Barbacid(1983). Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus bysingle point mutations. Nature 306:658–661.

51. J. E. Korkola and M. C. Archer (1999). Resistance to mammarytumorigenesis in Copenhagen rats is associated with the loss ofpreneoplastic lesions. Carcinogenesis 20:221–227.

Rat Models of Premalignant Breast Disease

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