Mammary Tumors of the Rat" a Review

CANCER RESEARCH

VoLvm~ 19 DECEMBER 1959 NUMBER 11

Mammary Tumors of the Rat" A Review

R. L. NOBLE AND J. H. CvT'rs

(Dept. of Medical Research, Collip Laboratory, University of Western Ontario, London, Canada)

Mammary tumors in the rat have been studied ORIGIN AND CHARACTERISTICS in detail by only a few groups of workers. There is, A. SPONTANEOUS MAMMARY TUMORS however, a considerable amount of information on 1. Incidence.--Spontaneous tumors in rats al- the subject contained in isolated papers and scat- lowed to live their normal life span are relatively tered throughout the scientific literature. Appar- common, and in large series of animals from 10 to ently, no previous attempt has been made to re- 60 per cent may show tumors of some kind (13, view this field of interest. In the past, the attention 119). The proportion of these which were confined of geneticists, virologists, endocrinologists, and to mammary tissue has varied in different reports biologists has been focused on the mammary tu- and with different strains of rats; however, mam- mors of the mouse, and a vast literature has ac- mary tumors have been the common type of tu- cumulated in which these tumors are characterized mor affecting the over-all tumor incidence. In the very exactly. The rat has been somewhat neglected August strain of rats 30 per cent of all tumors were in this concentration of effort on the smaller spe- of mammary origin, whereas in the Copenhagen cies, although it would appear worthy of greater strain only 5.6 per cent were similarly situated and more serious attention. The need for a tabula- (13). In another large series of rats of mixed tion of rat data may be appreciated from the ap- strains, 18 per cent of 521 spontaneous tumors pearance in 1954 of a paper in Science, containing were located in the breast (9). Of a group of 150 no references, entitled "Successful Transplanta- female Sprague-Dawley rats with an average life tion of an Apparently Benign Neoplasm." A rat span of 760 __+_21 days, mammary tumors ac- fibroadenoma apparently was transplanted suc- counted for 95 per cent of the total tumors found cessfully. This achievement should not have been and were present in 54 per cent of the animals. In a unexpected in view of Loeb's report in 1902 of group of 40 males, 12.5 per cent showed tumors of autotransplantation and in 1916 of homoiotrans- which only two were of mammary origin, giving a plantation of similar tumors in rats. general average of 88 per cent for both sexes. In Rat mammary tumors have many points of in- two colonies of rats of the Wistar strain mammary terest and differ from those of the mouse in many tumors accounted for 77 per cent of all tumors. In ways. Histologically, both benign and malignant females 281 of 839 (68 per cent) showed mammary tumors occur spontaneously in the rat, or may be tumors, whereas 42 of 129 males (32 per cent) had similar tumors (119). It is apparent that from 6 to induced by a number of means. No viral factors 77 per cent of all tumors occurring spontaneously have been demonstrated, and most rat tumors are in rats may be of the mammary gland and that curiously sensitive to hormone manipulation. females may show a ratio of approximately 6 to 1 Their possible value in chemotherapeutic screen- when compared with males. ing of hormone derivatives has only recently re- 2. Malignant tumors of epithelial or mesodermal ceived attention. All these considerations will be origin.--Malignant, spontaneous mammary tu- pointed out in the following review. mors in the rat have been classified as adenocar- Received for publication June 11, 1959. cinoma, solid carcinoma, cystadenocarcinoma, 1125

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. 11~6 Cancer Research Vol. 19, December, 1959 papillary carcinoma, and, rarely, squamous-cell mors. Similarly, Bullock and Curtis (9) examined carcinoma. Fibrosarcomas are more frequently en- 94 tumors of the breast and noted eight adenomas, countered, and mixed tumors combining adeno- one adenolipoma, and 78 fibroadenomas or fibro- carcinoma and squamous-cell cancer have been mas (93.4 per cent benign tumors). In 150 described. Thus, of 41 malignant spontaneous tu- Sprague-Dawley female rats, 90 per cent of 7~ mors encountered in a series of rats of mixed breast tumors were classed as benign--one adeno- strains, s per cent were classed as of epithelial ma, 46 adenofibromas, and eighteen fibromas (16). origin (seven adenocarcinoma, one squamous-cell Curtis, Bullock, and Dunning (13), after examin- carcinoma, and two adeno-acanthoma), 12 per ing over 30,000 rats from seven distinct strains, cent as mixed tumors (three sarcoma with adeno- found a total tumor incidence of 9.6 per cent, and, carcinoma and two with added squamous-cell car- of these, mammary fibroadenoma contributed cinoma), and 59 per cent as fibrosarcoma (28). 12.8 per cent. Of the 6410 female rats reaching an Bullock and Curtis (9), in examining 94 spontane- age of 14 months (considered to be the minimum ous breast tumors occurring in 489 rats of mixed time of appearance), 0.9 per cent showed benign origin, noted two cystadenocarcinoma with squa- fibroadenomas. Of 219 female Wistar rats bearing mous epithelium, two carcinosarcomas, and three mammary tumors and averaging 650 days of age, adenomas exhibiting sarcomatous change. Dun- 206 were classed as benign tumors. The growth ning and Curtis (23), in a study of 15,625 rats de- rate of the spontaneous fibroadenomas varied rived from twenty generations of pure strain Co- greatly. Of twenty tumors, eleven increased 2-10 penhagen 2331 and Fischer 344 rats and their times their original volume in 90 days (119). reciprocal F1 and backcross hybrids, also observed Metastases have not been observed. 82 mammary tumors. Of these, only two were 4. Factors influencing incidence of spontaneous malignant: one was an adenocarcinoma and one a tumors.- fibrosarcoma. Age: The age of the rat, as might be sus- In 149 mammary tumors arising in random- pected, influences the incidence of spontaneous tu- bred Albany strain of hooded rats, only one adeno- mors found at any time. Studies in which the ani- carcinoma was found (8, 144). In 468 rats of mals have been allowed to live their normal life Wistar origin exhibiting 273 mammary tumors, span have been the most informative. It was noted eleven mammary adenocarcinomas and two sar- that 80 per cent of fibroadenomas developed in comas were noted (119). Similarly, of 94 spontane- female Wistar rats between 64 and 128 weeks of ous tumors of the breast occurring in 489 rats only age, whereas carcinomas occurred somewhat ear- two were classed as cystadenocarcinoma and two lier in life (119). In the Albany strain of rats, be- as carcinosarcoma (9). Probably the highest inci- nign tumors arose chiefly between 52 and 88 weeks dence of spontaneous adenocarcinoma of the of age, although three tumors were noted in rats breast has been reported in 150 female rats of the only 20 weeks of age (144). In a comparison of Sprague-Dawley random-bred strain. Over an seven different strains of rats, fibroadenomas were average life span of 760 + 21 days, mammary tu- encountered in 0.9 per cent of 6410 females reach- mors were found in 72 rats (54 per cent) and, of ing 14 months of age (13). these, seven (10 per cent) were classed as adeno- Sex: As may be noted from the preceding dis- carcinoma. The over-all incidence of carcinoma of cussion, the sex of the rat may markedly influence the breast in these female rats therefore was 4.6 the occurrence of mammary tumors. Female ani- per cent. On the other hand, a smaller group of 40 mals were much more predisposed to this type of male animals of the same strain developed only tumor, although all types of tumors have been en- two mammary tumors (16). Metastases from spon- countered in males. The relative frequency of taneous malignant tumors have occasionally been mammary tumors has been low in males, possibly noted (8, 28). from 1 to 6 per cent of all mammary tumors. 3. Benign tumors of epithelial or mesodermal While exact experiments are not available, the origin.--Benign spontaneous mammary tumors implication is strong that the cause of the higher constitute a much larger group than do the preced- incidence in females is related to the influence of ing. Adenoma and adenolipoma have been de- the sex hormones. As will be seen later, estrogens scribed occasionally, but fibroadenoma and fibro- and progesterone may stimulate growth or be es- ma are apparently the most common tumor. Of the sential for successful takes of transplanted spon- 82 mammary tumors found in 15,625 rats pre- taneous mammary tumors, particularly the fibro- viously referred to (I~8), two were adenomas, two adenomas. On the other hand, it may be noted that adenolipomas, five fibromas, and 21 fibroadenomas mammary tumors induced by sex hormones are ba total of 93.7 per cent benign mammary tu- usually multiple, and practically all are classed as

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. NOBLE AND CuTTs--Mammary Tumors of the Rat: Review 1127 carcinoma. Some of the earlier workers were im- has been described as being discovered in 1938 on pressed with the possible role of a disturbed hor- the lower abdomen of a pregnant albino rat. It ap- mone balance as a cause of spontaneous tumors in parently regressed in size during lactation but the rat (70, 96) and believed that forced breeding grew rapidly again after the young were weaned. led to a higher incidence of tumors (2, 83). On the Histologically, it was an adenocarcinoma with a contrary, a high incidence of benign mammary tu- large proportion of connective tissue elements. mors in the Albany hooded strain of random-bred The tumor, during successive transplantation, has rats was believed to be related to a decreased fer- lost the original organized epithelial element and tility of unexplained cause (8). :Further studies at- now appears as an highly anaplastic growth (35). tempted to develop an inbred line of Albany rats The behavior of the transplanted tumor has been with a higher incidence of tumors and to determine studied by many workers. "Takes" have been ob- the endocrine imbalance responsible (144). tained readily in up to 96 per cent of rats of either Diet: A few papers of considerable interest have sex. The growth rate was rapid and reproducible, presented evidence that the diet, particularly the particularly on intramuscular implantation. It lipide content, may definitely affect the incidence could be transplanted to rats of most strains, and of mammary tumor development. Benson, Lev, it would grow, at a somewhat slower rate, even in and Grand (4) noted that the addition of 30 per the wild rat (137, 186). The tumor has been widely cent olive oil to a chow diet increased the incidence used for the screening of tumor inhibitors (140), of fibroadenoma in Sprague-Dawley female rats and it may show limited hormone responsiveness from 7 to 38 per cent by the time they were 28 (107). months of age. Groups of 43 controls and 139 oil- Eisen (36, 38) has described the transplantation fed animals were compared. No tumors appeared of mammary carcinoma in the August strain of before 18 months of age. Similarly, an incidence of inbred rats, and his observations may be consid- 57 per cent in 150 control Sprague-Dawley females ered as typical of other tumors of this type. Three was increased to 80 per cent in 100 animals fed a carcinomas arising in females of 335-553 days of diet containing 10 per cent corn oil (16). Diets con- age and one tumor in a male 748 days old were de- taining purified casein (118) or a low protein con- scribed. Transplantation was effected in 58-78 per tent (75) also have been found to lead to a higher cent of rats of the August strain. Age, sex, preg- incidence of breast fibroadenoma. In the latter nancy, or lactation did not influence tumor growth. paper, however, it may be noted that the low pro- The growth rate was rather slow. The tumors be- tein diet contained about 15 per cent fat from the came palpable in 3-3 weeks, measured 3-5 cm. in addition of lard and vegetable oil, whereas the diameter by 8 weeks, and killed the host in 3--4 control chow diet would contain less than 5 per months. Metastases were not observed. Intraperi- cent fat. In view of the preceding reports it seems toneal injection was followed by papillary nodular likely, therefore, that the higher fat content of the development on the omentum, whereas intrave- experimental diet may have been the major factor nous inoculation led to lung deposits. No change in contributing to the higher tumor incidence. the morphology or growth pattern was found over a 3-year period, in a comparison of the 3d and 37th B. TRANSPLANTED MAMMARY TUMORS transplant generation of one tumor, R3426. Simi- OF SPONTANEOUS ORIGIN larly, the moderate number of takes (38 per cent) 1. Malignant tumors.--Epithelial origin. The in related strains was unchanged, as was the occa- rarity of the occurrence of spontaneous malignant sional take (2 per cent in seven alien inbred tumors has allowed only a few papers describing strains). Growth of the transplant in rats of alien their characteristics. These, however, have been strains, when it did occur, was at a reduced rate complete studies from which generalizations can and associated with increased necrotic changes be made for the behavior of this type of tumor (38). Dunning, Curtis, and Maun (28) described a when propagated in pure strains of rats. Trans- mixed tumor, R2572, with adenocarcinoma and plantable mammary tumors of various origin in squamous-cell carcinoma components, which arose the rat have been included in the survey by Dun- in an 849-day-old A • C female rat. This re- ham and Stewart (19). Undoubtedly, the most mained as a mixed tumor through four transplant widely known transplanted mammary tumor of generations, but subsequently the two components the rat has been the Walker carcinosareoma 256. were successfully dissociated and maintained sepa- Because of the wide use of this tumor for purposes rately as a pure adenocarcinoma and an osteo- beyond the scope of this review, no attempt has ehondrosareoma. been made to summarize the literature. It is pos- 3. Benign tumors.--In contrast to the few re- sibly pertinent, however, to note that the tumor ports of spontaneous malignant tumors main-

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. 11 ~8 Cancer Research Vol. 19, December, 1959 tained by transplantation, a large number of stud- adenomas are affected by estrogens. These hor- ies have been made on benign tumors. Also in con- mones usually have been found to shorten the trast is the tendency for fibroadenoma to alter, latent period for growth in male and female rats morphologically, on repeated transplantation and and to increase the number of takes following frequently to show a marked response to the ad- gonadectomy. Tumor growth stimulation has been ministration of, or lack of, various hormones. Be- noted by many workers, particularly in the early nign tumors may be transplanted readily as origi- generations of a tumor line (61, 70, 80, 97, 98, nally described by Loeb (8~-84). The best results 144), although negative findings have been re- have been obtained by transplanting relatively ported when older generations have been used (4~, large, multiple pieces or slices of tumors, rather 43, 72, 104). than cell suspensions, in rats of the strain of origin Evidence of morphological changes during of the tumor. From 64 to 95 per cent successful treatment has also been noted (68, 70). The inter- takes in large series of female animals have been pretation of the effects of estrogens on tumor described (61, 67, 80, 90). Usually, prolonged growth has been complicated by the demonstra- latent periods and slow growth rates have been tion by Millar and Noble (89, 91) that, although noted. The characteristics of transplanted tumors small doses of estrogens increased the growth rate, originating from different spontaneous tumors, larger doses caused a marked depression of tumor however, have shown great variation. These in growth. Stilbesterol or estradiol in doses from 1 to turn are probably related to the tissue element 10 #g. were stimulatory to early transplant genera- predominating in the original tumor, the frequent tions, but levels of 50-100 ug. arrested growth. A diminution in epithelial elements during successive similar biphasic effect on the growth response of transplantation, and the related variations in re- tumors has been presented by Huggins et al. (80). sponse to hormones. Some tumors, however, have It should be noted that the inhibition of tumor been described which, over many generations, growth by nonphysiological doses of estrogens is have retained a constant pattern of growth and associated with a marked reduction in body morphology. Examples may be found in the ade- growth. Before ascribing a specific action to the noma and adenofibroma, unchanged after trans- estrogen (or other inhibitory steroid) comparison plantation for many years in inbred Wistar rats must be made with control rats reduced to the (15), a transplantable adenoma which showed lac- same growth rate by dietary restriction (93). tation (63), in an adenolipoma (114), or the fibro- Progesterone has been reported to have no effect adenoma described by Huggins and collaborators on tumor growth (91), to inhibit the epithelial part in Sprague-Dawley rats (80) and used by others of the tumor (71), or to stimulate growth, particu- for quantitative studies (61). Others, working with larly when administered with estrogens (78, 80). benign tumors of different origin, have been im- Most reports agree that androgens suppress tumor pressed with alterations in growth, hormone re- development and growth (61, 6~, 69-71, 78) and sponsiveness, and morphology, over succeeding may increase the tendency toward fibroma and generations (68, 70, 90). The transition of the be- sarcoma formation (69-71, 97, 99). Cortisone and nign tumor to a carcinoma (1~9) or a sarcoma (41, cortisol have little effect on tumor growth but may 44, 9~, 113, 1~r following repeated transplanta- inhibit the number of takes (61, 91). Reference will tion has been frequently noted. be made later to the use of a transplanted fibro- Transplanted fibroadenomas may or may not adenoma for screening steroids for chemothera- (61, 80) show wide variation in their response to peutic activity. hormones, depending upon the individual charac- Some findings strongly suggest that pituitary teristics of the tumor studied. In addition, altera- hormones may affect directly the growth of trans- tion in response over successive generations may plantable fibroadenoma (79, 80, 91). From the occur (90, 107). Most transplanted tumors show a work of Huggins et al. (79, 80) it may be seen that, hormone dependence on female sex hormones and following hypophysectomy, tumor growth, al- initially will transplant with limited success or not though inhibited initially, eventually proceeded at at all into male rats or ovariectomized females (54, a slow but progressive rate. Estrogen and pro- 64, 67, 80, 90, 1~0). The growth rate of the tumor gesterone combination therapy caused only a may be reduced markedly by ovariectomy and still moderate acceleration in growth rate. With the further following adrenalectomy or hypophysec- additional treatment of growth hormone, the tu- tomy (61, 80). Pregnancy has been found to stimu- mor growth rate was restored to normal. Luteo- late the growth rate of transplanted tumors in trophic hormone (prolactin) could not be substi- many cases, but great variation has been reported tuted for growth hormone, and none of these hor- (40, 43, 45, 63, 7~, 8e, 84, 90, 1~0). Most fibro- mones alone had any effect on tumor growth (79).

Transformation of fibroadenoma to sarcoma none of the nineteen A • C line rats developed may take place during hormone treatment, and it mammary tumors. However, 9 per cent of ~1 has been noted that any injections which markedly Marshal (M-5r strain, ~8 per cent of fourteen slowed the growth rate favored sarcomatous trans- Buffalo strain, 64 per cent of ~ Sprague-Dawley, formation (69, 70, 93). With such transformation, and 6~ per cent of ~1 Osborne-Mendel strain rats the hormonal susceptibilities of the original fibro- developed mammary tumors. In smaller groups of adenomas were largely or completely lost (44, 93, male animals, the incidence was 0, 0, 0, 35 and ~6 11~). Growth was rapid, and transplantation per cent, respectively. Dunning, Curtis, and Mad- could readily be effected into rats of either sex sen (~6) also investigated the response of five in- (11~) and even into rats of different strains (67). bred strains including the Copenhagen, August, Treatment of rats bearing transplanted fibroade- and Fischer strains. The Fischer rats were the noma with 1,~,5,6-dibenzanthracene has been re- most susceptible, the Marshal and Copenhagen ported (15). In male animals no change was ob- rats were resistant, while the August and A X C served, but in females the growth rate of the tu- strains showed intermediate sensitivity. Bielschow- mor was slowed. No consistent morphological sky (5, 6) noted a striking difference in an inci- changes in the tumors were found. Sarcoma devel- dence of 60 per cent in Wistar strain females com- oped at the injection site. Millar and Noble ~ in- pared with only 4 per cent in a piebald strain after jected 9,10-dimethylbenzanthracene into slowly the feeding of r Sherman and Wistar rats growing, transplanted fibroadenoma. In two cases have been found to exhibit approximately the adenocarcinomas were induced in the benign tu- same susceptibility (11). The induction period for mor. This type of malignant change had never mammary tumors with the more active carcino- been observed in control tumors. gens at dietary levels of 0.03 per cent was from r to 31 weeks, although this may be shortened by INDUCED MAMMARY TUMORS dietary measures (51). Mammary tumors may be induced readily in a As indicated in the preceding results, female high percentage of animals by a number of means. rats have been found to be consistently more sus- The mammary tissue of the rat appears to be ceptible than male animals (7, 11, 65, 184). Some highly susceptible to malignant change following observers have been impressed with the inverse treatment with aminofluorene and related com- ratio of the induction of liver and breast tumors in pounds, carcinogenic hydrocarbons, and various female rats by these compounds (135). Animals hormones. Curiously enough, induced tumors, in free from liver tumors may survive sufficiently contrast to the more common spontaneous tu- long to allow breast changes, but on the other hand mors, are generally carcinomas. Varying degrees of a possible failure to inactivate estrogens by an hormone responsiveness have been observed. altered liver function might be considered as an 1. Aminofluorene compounds.--The orally ac- etiological factor. When 2-AAF was administered tive carcinogen, 2-acetoaminofluorene, when fed at in doses of 100 rag. for 3 days it did not show a level of 0.03 per cent in the diet for periods longer estrogenic activity (5). The age at which feeding of than 95 days, was observed to induce cancer of the carcinogen was commenced had an important various tissues by Wilson, DeEds, and Cox (143). bearing on subsequent mammary tumor formation They reported three mammary adenocarcinomas, (7, 48). In a comparison of the first generation of of which one metastasized, in 39 female inbred three strains of rats (which began eating 2-AAF at albino rats of the Slonaker strain. This strain of 117 days of age), with the second generations, rats only rarely showed spontaneous tumors of the which started at 31 days, the incidence increased breast, and these were fibroadenoma. Many work- from 28 to 87 per cent, 64 to 91 per cent, and from ers have confirmed the induction of mammary 62 to 90 per cent, respectively (135). As might be cancer by feeding 2-aminofluorene (2-AF), 3- expected, the dose of the carcinogen consumed in acetylaminofluorene (3-AAF), or related com- the diet was important. Levels of 3-AAF below pounds. The incidence of mammary tumors thus 0.001 per cent were not effective in producing tu- induced has varied from 0 to 100 per cent owing to mors, whereas 0.004 per cent induced tumors in a number of controlling factors. Female animals 200--300 days. Feeding for periods of only 35 or 50 were more susceptible than males, and certain days lengthened the latent period for tumor devel- strains of rats showed a varied susceptibility. opment. Some mammary tumors were found in Symeonidis (135) studied five different strains of these studies (143). The oral feeding of carcinogens rats fed 0.03 per cent of 2-AAF for an average of of this type was not essential for the production of 350 days. In female animals surviving 120 days, mammary tumors, as these have been described 1 Unpublished observations. following treatment by injections (141).

The relative resistance of the male animal to the in the first eight generations the tumors tended to induction of these tumors has implicated the sex remain localized, thereafter they became more in- hormones in their production. Breeding females of vasive and faster growing. Subcutaneous, intra- low tumor strains have been found to be more muscular, or intravenous transplants were all suc- susceptible than virgin animals (135). Pregnancy cessful. Morphologically, the tumor changed from was associated with an increased growth rate of a carcinoma, with well defined acini and small the tumor. During lactation, however, growth was amounts of stroma, to a tumor showing areas of arrested, and some regression took place, although anaplasia, with sarcoma formation. Alterations in normal tumor growth was resumed when the growth rate after successive transplantation have young were weaned. Lactogenic hormone adminis- also been described in an induced tumor in AES tration was without effect (7). Gonad removal in rats (lg). The feeding of ~-AAF to rats bearing either sex markedly reduced the incidence of in- transplanted mammary fibroadenoma did not af- duced mammary tumors--one in eleven ovariec- fect their growth rate or induce malignant changes tomized compared with ~3 in 36 normal female (73). Metastasis from induced tumors occurred not Wistar rats; none in five castrated compared with infrequently, particularly if the animals were al- three in 41 normal males (5). The effect of hor- lowed to survive with large tumors (7, ~6, 14~). mones was extensively studied by Kirby (81) and The diet used for the rats during the experimen- Cantero, Stasney, and Paschkis (11, 134). When tal period may affect the incidence of mammary Sherman or Wistar rats fed ~-AAF were used, a tumors induced by ~-AAF (47). It would appear mammary tumor incidence of 30 per cent in fe- that a lowered incidence may be associated with a males and 0 per cent in males was observed (11). restricted caloric intake and that ordinary chow Treatment with 0.1~5 mg. of estradiol dipropio- diets may not be entirely adequate for maximum nate 3 times weekly had little effect on the incidence tumor production, or may exert a protective ac- in females, but allowed 6 per cent of males to de- tion. Engel and Copeland (51) noted, in two velop tumors. The administration of 0.5 rag. of strains of rats, that the use of purified diets al- testosterone propionate, 3 times a week, prevented lowed a shorter induction time and higher tumor any tumor induction in female rats. On the other incidence when compared with rats eating stock hand, administration of progesterone (0.5 mg. on diets. They believed the stock diets exerted a pro- the same dose schedule) increased the tumor inci- teetive action against tumor formation. Others dence in female rats to 85 per cent, and the tumors (137) have supported this view. Dietary riboflavin showed a more rapid growth rate. Progesterone did had little effect (49), but a low fat or high protein not enhance tumor formation in males or in ovari- level reduced the tumor incidence (141). A simple ectomized females. Chorionic or pregnant mare restriction of caloric intake, irrespective of fat con- serum gonadotrophin did not affect mammary tu- tent, was later shown to be associated with re- mor incidence (11, 134). Kirby did not detect any duced induction rate of mammary tumors (50). effect of androgens or estrogens on tumor induction Extensive ehemica! studies have been reported (81). on series of compounds related to ~-AF and s In many cases the tumors induced by amino- AAF. Although the primary motive of the experi- fluorenes were found to be multiple and in differ- ments was not concerned with mammary tumors, ent breasts of the rat. In most reports the mam- it may be noted that many compounds showed the mary tumors were classed as adenocarcinoma, with capacity to induce typical mammary adenocar- an occasional squamous, ductal, or papillary-cell cinoma (66, 74, 94, 103, 1~3, 124). The feeding of carcinoma, carcinosarcoma, or fibroadenoma (6, 7, hexanitrophenylamine to Wistar rats was believed ll, 74, 10g, 103, 148). In one report black-hooded to cause an increased incidence of multiple breast random-bred rats showed a predominance of fibro- fibroadenoma (138). adenomas (1~1). Transplantation of primary in- 2. Carcinogenic hydrocarbons.- duced tumors has been reported by several work- a) Subcutaneous Injection: The injection of ers. In two cases, more slowly growing adenocar- carcinogenic hydrocarbons in the rat typically cinomas required estrogen treatment of the host leads to malignancy close to the site of injection. for successful transplantation, although a more The injection, into areas of mammary tissue, of rapidly growing tumor arising in a male was ap- 1,~,5,6-dibenzanthracene, 3,4-benzpyrene, or 3- parently hormone-independent (5). The use of methylcholanthrene, in solutions of warm paraffin litter-mates was required for successful transplan- or wax, has given rise only very occasionally to tation in one case (148). With inbred Buffalo strain carcinoma of the breast (24, 25). Geschickter (57) rats, a transplanted tumor (No. 1643) was main- inserted pellets made of 1,~,5,6-dibenzanthracene tained for over twelve generations (102). Whereas and 3,4-benzpyrene into the mammary tissue of

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. NOBLE AND CuTTS---iammary Tumors of the Rat: Review 1131 male and female rats. Tumors were found in nearly DMBA was the only effective carcinogen tested. all rats in from 260 to 418 days, but these were all Negative results were obtained under comparable sarcomas, and the adjacent mammary tissue was conditions with 1,~-benzanthracene, g0-methyl- of normal microscopic appearance. There is little cholanthrene, 1,3,5,6-dibenzanthracene, and p-di- evidence of a general systemic action on the breast methylaminoazobenzene. In a later paper (60) from these forms of administration, although the emulsions containing 0.25 mg. of DMBA were appearance of sarcoma locally may reduce the life given by three intravenous injections over 1 week span of the animal below the latent period for and repeated 3 weeks later. In female Sprague- mammary tumor development. Dawley rats an incidence of 80 per cent mammary b) Intramuscular injection: During attempts to tumors was noted in 48 weeks. The addition of a modify the local action of the carcinogen, 7,12- total of 0.6 rag. of a-estradiol to the injection mix- dimethylbenz[a]anthracene (DMBA) (formerly ture led to a higher incidence and earlier appear- referred to as 9,10-dimethylbenzanthracene) was ance of tumors. Similarly, diethylstilbestrol, added mixed with cholesterol before intramuscular injec- in a total dose of 5.28 mg., caused an increase in tion. Under some conditions the induction of local the total number of tumors so that 90-94 per cent sarcoma was inhibited or markedly delayed, and of the rats showed tumors in 19 weeks. The estro- in these rats a high incidence of mammary tumors gens administered alone in 5-6 times the dose did developed. Rats of a black-hooded random-bred not induce tumors after 25 weeks. The tumors strain received a single injection, intramuscularly which developed were mostly adenocarcinoma or into the thigh, of a solution of 5 rag. of DMBA and adenoma (92 in 1~7 rats), although sixteen fibro- 10 mg. of cholesterol in 0.5 cc. of sesame oil. After adenomas also were found. Scholler (1~5, 126), 7 months, twenty female rats had developed mam- using a similar intravenous technic for Wistar rats, mary tumors, frequently multiple. Histologically, induced 89 per cent tumors in 14 weeks, the me- all were typical benign adenofibromas. Trans- dian time of appearance being 59 days. Pregnancy plantation was readily effected into rats of either and lactation did not alter the incidence or growth sex of the same strain (106). It is curious that tu- rate. Physiological and pharmacological doses of mors induced by this means should be adeno- estradiol, stilbestrol, progesterone, and estradiol fibroma, since, as will be seen subsequently, mam- plus progesterone were similarly without demon- mary tumors induced by carcinogenic hydrocar- strable effect. Transplantation of induced tumors bons have, with few exceptions, been adenocar- was rarely successful and was not influenced by the cinomas. The strain of rats used was the same as treatment of tile host. Recipients included rats of that found by others to develop fibroadenoma both sexes and of various age groups, castrate after 3-acetylaminofluorene feeding, to contrast males, pregnant rats, and rats conditioned by hor- again to the usual adenocarcinoma (121). On the mone or DMBA treatment (126). other hand, the long induction period, up to 7 d) Oral administration: Shay and collaborators months, is in contrast to that following the in- (131-33) first observed the development of mam- travenous injection or gastric installation of car- mary tumors in rats receiving repeated gastric in- cinogenic hydrocarbons and may favor the devel- stillations of methylcholanthrene in oil, starting opment of histologically benign tumors. Intra- when the rats were 60-80 gm. in weight. In female, muscular injections of DMBA in oil alone in the random-bred Wistar rats receiving 2 rag. of car- same black-hooded strain was followed by the de- cinogen daily, 6 days every week, 83 per cent of the velopment, near the site of injection, of adenocar- treated rats developed tumors. Male rats and cinoma of the breast in four of 31 female rats in ovariectomized females were much less suscep- 4-5 months. In nineteen hypophysectomized ani- tible. In sixteen of eighteen female rats, tumors mals similarly treated, no mammary tumors were developed in an average of 195 days. Tumors in six observed (111). of fourteen males did not appear until 386 days, c) Intravenous Injection: Repeated intravenous and in females spayed when immature only three injections of carcinogenic hydrocarbons were first of eight showed tumors at 466 days. The earliest shown by Geyer and collaborators (59) to be fol- tumors were observed after 150 days, and multiple lowed by the development of mammary tumors in growths occurred frequently. Occasionally, metas- rats. Using Wistar or Sprague-Dawley rats of 150 tases to the lung occurred. When steroid pellets gin. in weight, they administered twelve weekly implanted in the subcutaneous tissue were used as injections. In female rats, ~4 adenocarcinomas or a form of therapy, it was believed that progester- adenomas and one fibroadenoma were noted, an one and testosterone decreased the occurrence of incidence of 32 per cent. The tumors were often tumors in females, whereas injection of follicle- multiple and invaded the surrounding tissues. stimulating hormone increased the incidence in

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. 1182 Cancer Research Vol. 19, December, 1959 males. Adenocarcinomas were most frequently en- form of prophylaxis against this type of induced countered in female rats, but fibroadenomas were cancer in the rat. Dao and colleagues (14) in studies more common in males treated with estradiol. It using orally administered methylcholanthrene was believed that the hormone balance maintained have noted an increase in size and number of the by treatment influenced the type of tumor which induced mummary tumors during pregnancy in developed. Huggins and collaborators (76, 77) Sprague-Dawley rats--an increase from 50 per have achieved more rapid induction of tumors in cent in controls after 65 days to a 90 per cent in- 100 per cent of Sprague-Dawley rats, by the daily cidence. Following parturition all tumors were oral administration of 10 mg. of 3-methylcho- seen to regress immediately and some disappeared lanthrene in sesame oil. In a large series of rats, completely in 1-8 weeks. treatment was started at 50 days of age, and all e) Skin painting: Carcinogenic hydrocarbons animals had developed tumors in 60 days. If 10 have been applied to the skin of rats by Orr and rag. was given daily for only 10 days, 100 per cent colleagues (115-17), and it was noted that this was of animals developed tumors by 3 months. Three followed by mammary tumor induction. Rats of doses/week were apparently as effective as daily three different strains were painted with a solution administration. DMBA given orally at a dose level of either 0.5 or 1.6 per cent DMBA. Mammary tu- of 1.0 mg. daily induced tumors in all of nine rats mors developed in 73 per cent of animals in both by 79 days. Of 680 induced mammary tumors, 678 groups. With the higher concentration of carcino- were classed as carcinoma and two as fibrosar- gen, there were more multiple tumors, and the coma. The normal mammary epithelium showed average time of appearance was 1~.6 weeks, where- increased epithelial proliferation. Metastasis was as with the lower concentration ~7 weeks was re- not seen, but infiltration of the tissue was frequent. quired for tumor induction. A single application of Most of the tumors studied showed hormonal de- a 1.6 per cent solution of DMBA in olive oil pro- pendency, since they decreased in size after ovari- duced six tumors in nine female rats after about 13 ectomy, and especially following hypophysectomy. months. Solutions of methylcholanthrene when ap- A few tumors showed greater autonomy and con- plied by the same technic failed to induce tumor tinued to grow after ovariectomy. Histologically formation in 15 months. Histologically, all the tu- these tumors showed areas of atrophy with other mors were adenocarcinomas except for two col- areas of progressive growth. Treatment with 0.1 lagenous and one fibroadenoma. One tumor was gg. daily of estradiol-17//~ allowed a normal inci- transplanted through three generations but exhib- dence of tumors in ovariectomized females. A dose ited a very slow growth rate. A study of the estrous of 1 gg. delayed the appearance of tumors: ~0 pg. cycle of the treated rats failed to show any indica- caused a further delay, and only one-third of the tion of estrogenic action of DMBA. In view of the animals developed tumors. Progesterone, at a dose other work which has been reviewed it would seem of 4 rag. daily, apparently accelerated the time of likely that skin painting with DMBA was followed appearance, whereas dihydrotestosterone, 1 rag. by general absorption of the carcinogen so that the daily, delayed the onset and inhibited tumor de- breast was influenced by a systemic, rather than a velopment. The stimulatory effects of progester- local action. one could be blocked by estradiol. The growth rate 8. Hormones.-- of established tumors e5 days after the cessation of a) Growth hormone: Evans and Simpson (5~) administration of the carcinogen was also studied. studied the action of prolonged administration of Tumors in control rats continued uninterrupted the growth hormone which was available in 1931 growth. Ovariectomy reduced the growth rate, but and first called attention to the induction of mam- this could be accelerated by 5 pg. daily of stil- mary fibroadenoma in rats of the Long-Evans bestrol. Hypophysectomy caused a marked slow- strain. Tumors appeared in ten of sixteen animals ing in growth, and dihydrotestosterone, 1 or ~ rag. treated over a 16-month period. No tumors were daily, also reduced the rate of tumor growth. observed in similarly treated male rats. In a more Mammary cancer development was apparently recent report (100), highly purified pituitary prevented in hypophysectomized rats over an 8- growth hormone was administered for periods up month period (76). This finding and the similar to 485 days to female rats of the same strain. After observation that an injected carcinogen was not 380 days of treatment, commencing with 0.4 mg. effective in inducing mammary cancer in hypophy- daily and gradually increasing to 3.0 mg. of growth sectomized in contrast to intact rats (111) sug- hormone, eight of fifteen animals had developed gests that pituitary hormones may play an essen- mammary fibroadenoma or fibroma, in addition to tial role in tumorigenesis of the mammary gland of tumors of other organs. In a control group, three the rat. Pituitary removal may be looked on as a of fifteen rats showed similar mammary tumors.

Of these, two had an additional pituitary adeno- old rats, but only 90 days in those treated when s ma. It was noted that many of the treated animals months of age. In total, 202 tumors were induced showed a local or generalized development of the in 555 rats of either sex, ovariectomized or cas- mammary tissue. The tumors were frequently trated, and these were mainly carcinomas, al- multiple, and two rats each had eleven tumors. In though some fibroadenomas were noted. Neither a similar experiment using hypophysectomized progesterone nor testosterone treatment induced rats, no tumors were observed (101). It would ap- tumors. Dunning and colleagues (32-34) found pear that in this strain of rats growth hormone that different strains of inbred rats showed differ- increased the frequency of fibroadenoma above ent susceptibilities to tumor development follow- that occurring normally. ing estrogen pellet imPlantation. The August line b) Estrogens: The continuous administration of 990 was the most susceptible but tolerated the estrogen to rats has been followed by a high inci- treatment with estrone pellets poorly. In the dence of mammary tumors, usually carcinoma. It A X C line 9935, 25 per cent of males and 18 per may be noted that secondary changes in the secre- cent of females developed tumors. In Fischer line tion of hormones by the pituitary gland and pitui- 344, 7 per cent of males and 16 per cent of females tary adenoma formation also occur following estro- were susceptible, whereas the Copenhagen line gen treatment. Pituitary hormones, therefore, may 2331 was completely resistant. Pellets made from be implicated in mammary tumor development, stilbestrol mixed with cholesterol were more effec- and mammotrophin hormone-secreting pituitary tive than those made from estrone alone. The tumors have been studied extensively by Furth A • C rats and August rats showed 80-85 per cent and collaborators. Although such transplanted tu- tumors, the Fischer 17-22 per cent, while the mors may cause a marked stimulation of mam- Copenhagen were again totally resistant. Multiple mary tissue in Fischer rats, only occasional evi- tumors were found more frequently after stilbes- dence of tumor formation has been observed under trol than after estrone treatment. Of 133 tumors the conditions of the experiments, which were not examined histologically, 111 were adenocarcino- primarily designed to induce tumors (55). The oc- mas, eleven adenocarcinomas and squamous-cell casional development of mammary tumors was carcinomas, six solid carcinomas, one interductal noted by some workers following the administra- carcinoma, and four were unclassified. The Copen- tion of estrogens (39, 86, 88). hagen rats showed a stimulation of mammary tis- Geschickter (56, 57), Noble, McEuen, and Col- sue by estrogens, but tumor formation did not take lip (110), and Nelson (105), on the other hand, ob- place. Nelson (105) observed 68 tumors induced by served that a high incidence of tumors could be in- estrogen in 103 rats of the Long-Evans strain after duced if estrogens were injected or, preferably, im- 300 days treatment. Metastasis occurred in 33 ani- planted subcutaneously as pellets. Additional mals. These tumors were classified as duct car- studies on the induction of mammary tumors by cinoma in 42 rats, adenocarcinoma in eight, com- estrogens have been reported by many workers bined duct and adenocarcinoma in thirteen, and (21, 22, 27, 29, 30, 32-34, 57, 58, 85, 86). Geschick- carcinoma simplex in five. Others have found a dif- ter (56, 57), using an inbred strain of albino rats, ferent susceptibility in inbred and random-bred' produced mammary tumors in all animals by the rats (85), but the Copenhagen line appears to be injection or pellet implantation of estrogens. The the only resistant one described. This form of in- time of appearance of the tumors varied with the duced carcinoma apparently may metastasize dose. Injection of 30 pg. daily, of estrone required readily (105), but in some reports only occasional 600-700 days for the appearance of tumors, where- secondary deposits have been noted (37, 57, 58, as 200 ~g. daily required only 150-200 days. Pel- 108). Transplantation has not been studied exten- lets of 3-10 mg. stimulated tumor development in sively, but difficulty has been experienced with 50-200 days, one tumor being noted after only 21 random-bred rats (108, 109). days. Daily injections of 100 ~g. of estradiol re- The influence of diet on tumor induction by quired 185 days for tumors to appear; the same estrogens has been extensively studied by Dun- dose of stilbestrol required 200 days, and, likewise, ning, Curtis, and collaborators (22, 29, 30). Tu- estrone required 875 days. The tumors were main- mors were induced in the A X C rat by the sub- ly of comedo type, duct and scirrhous carcinoma, cutaneous implantation of cholesterol-diethylstil- frequently multiple, and many metastasized. In a bestrol pellets. Caloric reduction by 26-38 per cent later paper (58) it was shown that the time of ap- of the control animals did not reduce the tumor in- pearance of the tumors was inversely proportional cidence in 67 rats surviving 180 days. Of these, 87 to the age of the rat at the start of the experiment. per cent showed multiple mammary cancers, as Tumors required 293 days to develop in 1-month- 236 gross tumors and 337 microscopic tumors. The

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. 1134 Cancer Research Vol. 19, December, 1959 latent period for the appearance of tumors was, tumors induced by administration of sex hor- however, extended to 400 days in the low-calorie mone" (145). On the other hand, electron micro- group, from a control time of 300 days. Increasing scopical examination of a breast carcinoma (T4~3), the fat content of the diet tended to reduce the induced in a rat following the intraperitoneal in- latent period and to accelerate the growth rate of jection of radioactive gold, failed to show any evi- the tumors (39). The tryptophan content of the dence of virus-like particles (1~8). The most con- diet was also considered to be important (~3, 80). vincing and direct experiments were reported by The addition of 1.4 per cent DL-tryptophan in- Dunning, Curtis, and Madsen (31, 37). These ob- creased the incidence of tumors from 77 to 100 per servers used inbred Copenhagen rats as a strain cent, the control rats having a total of 51 tumors totally resistant to estrogen-induced mammary compared with 79 in the experimental group. The cancer, and the August and A X C strains as sen- treated group survived only 316 days compared sitive ones (as previously described). They then with 868 days for the controls. Of the 161 tumors studied the induced tumor incidence in reciprocal induced, 107 were papillary cystadenocarcinomas, FI hybrids between resistant and sensitive strains. 44 adenocarcinomas and solid carcinomas, nine In a large series of animals the hybrids from both showed varying amounts of squamous-cell cancer, series showed an equal incidence and similar latent and one was unclassified (30). A further study, periods of tumor development. They concluded, using a purified diet deficient in tryptophan, indi- "If the susceptibility of the hybrid to the induced cated that deficient rats did not show as high an neoplasms was controlled by a factor transmitted incidence of estrogen-induced mammary tumors in the milk of the susceptible parent (such as ob- (56 per cent) as did controls (85 per cent), but sur- tained in some inbred strains of mice) the percent- vival times were considerably reduced on the de- age of individuals with induced mammary cancer ficient diet (33). among the progeny of the mothers of the suscep- The hormonal dependence of tumors in experi- tible strain should have equalled that obtained in mental animals was first described in 1941 with rats of the susceptible parental strain and should estrogen-induced tumors in rats (108). The sub- have been zero or relatively low among the prog- cutaneous implantation of estrone pellets into ran- eny of mothers from the resistant strain." Since no dom-bred black, hooded rats was followed by de- differences were found in the two groups of hy- velopment of adenocarcinoma in 38 of 49 rats, the brids, no evidence was found to indicate a ma- first tumor appearing after 3~6 days. In four rats, ternally transmitted etiological factor. It was also the surgical removal of the pellet containing the pointed out that, unlike mammary cancers in hormonal stimulus was followed by a rapid regres- agent-free mice, the rat tumors showed very little sion of all mammary tumors. Later reimplantation variation in morphology. Of a total of 351 tumors of pellets into two of these anita als led to the reap- examined, 343 were papillary cystadenocarcino- pearance of mammary tumors. Progesterone treat- mas. In a different type of experiment Ambrus and ment caused a slowing in growth of the induced Harrisson (1) inoculated Sherman rats with an ex- mammary tumors in four rats. These experiments tract of mouse mammary glands containing the more recently have been repeated and confirmed milk agent. There was no effect on the rat off- (109). Nelson (105) noted tumors induced by es- spring. Normal rats were allowed to foster-nurse trone injections, in seven of twelve animals, which young virus-free mice. These remained free of apparently continued to grow after the cessation mammary tumors so that an effective agent for the of injections, but residual amounts of estrogen mouse was not present in rat milk. might be expected to remain in the animal for a considerable period of time. MAMMARY TUMOR-HOST RELATIONSHIP IS A VIRUS FACTOR IMPLICATED A few disconnected observations on the re- IN RAT MAMMARY TUMORS? sponse of the host to mammary tumors have been Despite the extensive observations on the role reported. Dickinson, Begg, and Millar (3, 18) of a viral agent in spontaneous mammary tumors noted that liver catalase values were essentially in certain strains of mice, only a few similar studies normal for Sprague-Dawley rats bearing trans- have been made on rats. These show little evidence planted fibroadenomas. However, the sarcomatous that a virus plays any role in the occurrence of transformation of similar transplanted tumors was mammary tumors. One reference to a translation followed by a 50 per cent loss of liver catalase ac- from a Russian paper states, "a group of Russian tivity. This change was not explained by an altera- workers found a virus-like agent in rat mammary tion in liver size or growth rate of the tumor. They

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. NOBLE AND CuTTs--Mammary Tumors of the Rat: Review 1135 also noted that the fibroadenomas caused a sig- CHEMOTHERAPEUTIC STUDIES nificant degree of anemia in the host. Field (53) Recent interest has centered on the possible use observed a reduction in the prothrombin time of of a transplantable mammary tumor of the rat Sprague-Dawley rats bearing spontaneous fibro- which might serve for chemotherapeutic studies. adenomas. Increased size of the liver and pituitary Some type of hormonally sensitive tumor would be gland was also associated. Following tumor re- required to screen compounds of steroid structure. moval, all values returned to normal. The cathep- As indicated in this review, certain tumors, espe- tic activity of the liver and kidneys has also been cially the fibroadenoma and the carcinoma in- found to be increased in the Buffalo strain of rats duced by orally administered carcinogens, have bearing a transplanted aminofluorene-induced tu- been used by Huggins and collaborators for assay mor (87). The spleens of such animals showed in- purposes. The tumors which appear suitable for creased extra-medullary hematopoiesis. Rats of further consideration will be briefly summarized. the August strain bearing transplanted adenocar- Eisen (37) originally noted that estradiol benzoate, cinoma R 2426 were offered a free choice of various 0.166 pg. twice weekly, caused an inhibition of diets (10). No effects of diet were noted on tumor growth rate of adenocarcinoma R 2426 carried in growth, nor did the tumor-bearing rats select any the August strain of rats. Caloric restriction of diet preferentially. After 6 weeks' growth the controls, however, resulted in similar alteration in transplants averaged 11.05 gm. in weight per rat. the tumor growth rate. Testosterone had no effect. The radiosensitivity of the preceding tumor has Dunning (20) more recently has used the same been studied by Eisen (38). The tumor proved to tumor and found that estrone, 20 pg. daily, and be considerably more resistant than the Walker progesterone, 1 rag. daily, had no effect on tumor tumor in Wistar rats (17). Irradiation of 5,500 r in growth; deoxycorticosterone acetate, 6 rag. daily, vitro was required to abolish proliferative capacity had a slight stimulatory effect. Cortisol, 2-4 rag. of R 2426. With less irradiation, some tumors grew daily, and testosterone, 2 mg. daily, caused a sig- slowly after a prolonged latent period. Subsequent nificant reduction in the growth rate. The estro- transplantation showed a growth rate like the gen-induced carcinoma, which showed such a strik- original, so that no induced changes in the cells, ing dependence on estrogens for continued growth, altering their growth rate, had occurred. Treat- has not yet been used for steroid assay purposes ment of growing tumors with 12,000 r did not al- (108). Unless such tumors would retain their hor- ways cause complete regression. Heiman (67) com- monal dependency after transplantation, possibly pared the sensitivity to x-radiation of fibroadeno- in pure strain rats, their usefulness would be lim- ma and of fibrosarcoma arising from them. He ited. noted that 5500 r and 8500 r, respectively, were The benign fibroadenoma studied by Huggins required to effectively destroy the cells. et al. (78, 80) showed a consistent growth rate over Two reports concern a study of metastasis. In successive transplant generations and has been one (31) the transplantable adenocarcinoma found to be particularly hormone-sensitive. R 2426 was successfully implanted into the pros- Growth stimulation followed treatment with small tate gland. Metastasis was not noted after estro- doses of estrogen and progesterone. Its particular gen stimulation, although induced prostatic car- value, however, was for the assay of the growth- cinoma in the strain of rats used regularly metas- inhibiting properties of many compounds of the tasized to bone. Wallace (139) studied the metas- androstane series. Huggins and Mainzer (78) tasis from a group of sarcomas, all of which origi- found 2-a-methyl-dihydrotestosterone to be the nated from a transplantable man, mary fibro- most active inhibitor tested and presented evi- adenoma. The occurrence of spontaneous metas- dence indicating that the property of inhibiting tasis varied with the different tumors observed and tumor growth did not necessarily parallel andro- could be increased by mechanical means. Intra- genic activity. This tumor has recently been stud- venous inoculation was followed by metastasis but ied by others (61) and has also been used to screen only in the sublines which were capable of spon- steroids of the androstane series (62). The quanti- taneous metastasis. Either the tumors possessed tative method described appeared to yield consist- different intrinsic properties to metastasize, or the ent results and was readily performed. With some resistance of the host was differently affected. compounds the antitumor action was again not A high content of estrogenic substance occurring related to androgenic activity. The potential dan- in transplanted mammary fibroadenoma in rats ger in using fibroadenomas for quantitative assay has been reported (72). Others, however, failed to procedures has been previously pointed out. Any confirm this observation (95, 144). tendency for alterations in growth or hormone re-

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1959 American Association for Cancer Research. 1136 Cancer Research Vol. 19, December, 1959 sponsiveness in progressive transplantations must 15. DAvis, J. H.; MURPHY, K. M.; and EMGE, L. A. Effect of be noted. Dibenzanthracene on Transplantable Mammary Adeno- fibroma of the White Rat. Am. J. Path., 17:93-102, 1941. Adenocarcinomas induced by 2-acetylamino- 16. DAVIS, R. K.; STEVENSON, G. T.; and BUSCH, K. A. Tu- fluorene have not been used for steroid assay pur- mor Incidence in Normal Sprague-Dawley Female Rats. poses, although, as has been pointed out, some Cancer Research, 16:194-97, 1956. tumors showed a marked stimulation by proges- 17. Departments of Physics and Radiotherapy--Royal Can- terone (11). cer Hospital. Brit. Empire Cancer Campaign, 31st Annual Report 1953, p. 62. Tumors induced by the oral administration of 18. DICKINSON, T. E., and BEGG, R. W. Liver Catalase Ac- carcinogenic hydrocarbons appear to fill most of tivity in RaLs Bearing Benign and Malignant Tumors. the requirements for quantitative assay studies Cancer Research, 11:244, 1951. (77). It has already been noted that these induced 19. DVNHAM, L. J., and STEWART, H. L. A Survey of Trans- plantable and Transmissible Animals Tumors. J. Nat. adenocarcinomas respond to the female sex hor- Cancer Inst., 13:1299-1377, 1953. mones, and inhibiting effects of an androgen have o0. DUNNING, W. F. Response of Some Isologously Trans- been demonstrated (77). The primary induced tu- planted Rat Neoplasms to Steroids. Ann. N. Y. Acad. mors could be used for assay purposes, or possibly Sc., 76: 696-704, 1958. further studies will show that transplants may re- ~1. DUNNING, W. V., and CURTIS, M. R. Incidence of Di- ethylstilbestrol-induced Cancer in Reciprocal F1 Hybrids tain hormonal responsiveness. 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Production of Tumors in Rats by 2-Amino- Exper. Biol. & Med., 38:338-41, 1938. fluorene and 2-Acetylaminofluorene. Failure of Liver Ex- 45. EMGE, L. A., and WULFF, L. M. R. Influence of Preg- tract and of Dietary Protein Level To Influence Liver nancy on Experimental Tumor Growth in White Rat: Tumor Production. Cancer Research, 7: 88-94, 1947. Volumetric Studies on Adenofibroma and Fibroma. West 66. HARTMANN, H. A.; MILLER, E. C.; MILLER, J. A.; and J. Surg., 42:45-54, 1934. MORRIS, F. K. The Leukemogenic Action of 2-Acetyl- 46. EMGE, L. A.; WULFF, L. M. R.; and TAINTER, M. L. Ef- aminophenanthrene in the Rat. Cancer Research, 19: fects of Dinitrophenol on an Experimental Sarcoma of the 210-16, 1959. White Rat. Proc. Soc. Expel Biol. & Med., 31:152-54, 67. HEIMAN, J. The Study of Benign Neoplasms of the Rat's 1933. Breast. Am. J. Cancer, 22:497-524, 1934. 47. ENGEL, R. W. Dietary Factors Influencing the Car- 68. ~. 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77. HUGGINS, C.; BRIZIARELLI, G.; and Sv'rToN, H., JR. 97. --.. The Effect of the Sex Hormones on the Growth of Rapid Induction of Mammary Carcinoma in the Rat and Transplanted Mammary Adenofibroma in Ratu. Am. J. the Influence of Hormones on the Tumors. J. Exper. Cancer, 38:21~-16, 1940. Med., 109:~5--42, 1959. 98. . Effect of Estrogens and Androgens on Growth of 78. HUGGINS, C., and MAINZER, K. Hormonal Influences on Mammary Fibroma in Rats. Proc. Soc. Exper. Biol. & Mammary Tumors of the Rat. II. Retardation of Growth IVied., 43: 270-72, 1940. of a Transplanted Fibroadenoma in Intact Female Rats 99. . The Transformation of Rat Mammary Fibro- by Steroids in the Androstane Series. J. Exper. Med., adenoma to Fibroma by Androgens. Cancer Research, 10w 485-500, 1957. 1:151-53, 1941. 79. HUGGINS, C.; TORRAI,BA, Y.; and CHARR, A. Endocrine 100. MOON, H. D.; SIMPSON, M. E.; LI, CI H.; and EVANS, Influences on Growth of a Benign Transplantable Mam- H. M. Neoplasms in Rats Treated with Pituitary Growth mary Tumor. Science, 123: 674, 1956. Hormone. III. Reproductive Organs. Cancer Research, 80. HUGGINS,C.; TORRALBA, Y.; and MAINZER,K. Hormonal 10: 549-56, 1950. Influences on Mammary Tumors of the Rat. I. Accelera- 101. . Neoplasms in Rats Treated with Pituitary tion of Growth of Transplanted Fibroadenoma in Ovari- Growth Hormone. V. Absence of Neoplasms in Hypoph- ectomized and Hypophysectomized Rats. J. Exper. Med., ysectomized Rats. Ibid., 11:535-59, 1951. 104: 5~5-38, 1956. 10~. MORRIS, H. P.; DUNN, T. B.; and D~BNrK, C. S. Two 81. KmBY, A. H. M. The Combined Action of 2-Acetomino- Transplantable Rat Carcinomas Induced ~ith N-Acetyl- fluorene and Sex Hormones in the Wistar Rat. Brit. J. ~-aminofluorene. J. Nat. Cancer Inst., 9:225-28, 1948. Cancer, 1: 68-79, 1947. 103. MORRIS, H. P.; VEUCT, C. A.; and WAO~ER, B. P. Car- 82. LOEB, L. 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MARK, J., and BISKIND, G. R. The Effect of Long Term Mammary Adenofibroma in the Rat by 7,12-Dimethyl- Stimulation of Male and Female Rats with Estrone, benz(a)anthracene. Proc. Canad. Fed. Biol. Soc., 1:38, Estradiol Benzoate, and Testosterone Propionate Admin- 1958. istered in Pellet Form. Endocrinology, 28:465-77, 1941. 107. ~--. Hormonal Regulation of Tumor Growth. Phar- 87. MAVER,M. E. ; DUNN, T. B. ; and G~co, A. The Cathep- macol. Rev., 9: 867--4~6, 1957. tic Activities of the Tumors and Tissues of Tumor-bearing 108. NOBLE, R. L., and COLLIP, J. B. Regression of Estrogen- Rats. J. Nat. Cancer Inst., 8:39-41, 1948. Induced Mammary Tumors in Female Rats Following 88. McEuEN, C. S. Occurrence of Cancer in Rats Treated Removal of the Stimulus. Canad. Med. Assoc. J., 44:1-5, with Oestrone. Am. J. Cancer, 34:184-95, 1938. 1941. 89. MILLAB, M. J., and NOBLE, R. L. A Study of the Factors 109. NOBLE, R. L., and C~rTs, J. H. 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150. ROBINSON, G. H., and GRAUER, R. C. Transplantable 138. SHAY, H.; HARRIS, C.; and GRUENSTEIN, M. Influence of Benign Adenomas in Rats. Am. J. Cancer, 10:184-90, Sex Hormones on the Incidence and Form of Tumors 1952. Produced in Male or Female Rats by Gastric Instillation 121. Ross, R. C.; SCARF, R. F.; and SKORYNA, S. C. Histo- of Methylcholanthrene. J. Nat. Cancer Inst., 13: 307-3~, genesis of Breast Tumors in Rats Receiving 2-Aeetyl- 1955. aminofluorene. Arch. Path., 55:173-80, 1953. 184. STASNEr, J.; PASCHKm, K. E.; CANTAROW, A.; and 1~2. RoussY, G.; GUERIN, P.; and GUERXN,M. Nouvelle ~tude ROTHENBVaa, M. S. Neoplasms in Rats with 5-Acetami- exp~rimentale des tumeurs mammaires transplantables nofluorene and Sex Hormones. Cancer Research, 7:856- chez le rat. La transformation des fibro-ad~nomes en 61, 1947. tumeurs complexes du sein. Bull. acad. de mrd., Paris, 185. SYMEONIDI~, A. Tumors Induced by 2-Acetylamino- 129: 417-23, 1945. fluorene in Virgin Breeding Females of Five Strains of 1~3. SCHINZ, H. R.; FRITZ-NIGGLI, H.; CAMPBELL, T. W.; and Rats and in Their Offspring. J. Nat. Cancer Inst., 15: SCHMID, H. Krebsbildung durch Aminofluorene und ver- 539-49, 1954. wandte KSrper. Oncologia (Basel), 8:233-45, 1955. 136. TALALAY,P.; TAKANO, G. M. V.; and HVGCINS, C. Stud- 124. SCRMXrtL, D., and MECKV., R., JR. Quantitative Unter- ies on the Walker Tumor. I. Standardization of the suchung der carcinogenen Wirksamkeit von 4-Aminostil- Growth of a Transplantable Tumor. Cancer Research, benen. Ztschr. Krebsforsch., 61: 23(}-39, 1956. 12: 834-37, 1952. 125. SCHON,ER, J. Practical and Theoretical Considerations in 137. TUBA, J.; RAW~NSON, H. S.; FRASER, M. S.; and JESKE, the Use of Induced and Spontaneous Mammary Tumors I. Hyperplastic Nodules in Rat Mammary Glands follow- in Cancer Chemotherapy. Ann. N.Y. Acad. Sc., 76:855- ing the Feeding of 4-Acetylaminobiphenyl. Canad. J. 60, 1958. Research, 31: 95-98, 1953. 138. VAN EscR, G. J.; VINK, H. H.; and VAN GENDEREN, H. 126. SCHOLLER, J., and CARNES, R. E. Preliminary Studies Influence of Hexanitrophenylamine on the Incidence of with Mammary Tumors in the Rat Induced by 7,1 ~ Neoplasms in the Mammary Tissues of Rats. Nature, Dimethylbenz(a)anthracene (DMBA). Proc. Am. Assoc. 180: 509-10, 1957. Cancer Research, 1: 343, 1958. 139. WAL~CE, A. C. The Occurrence of Metastases in a Group 127. SCHREK, R. A. A Quantitative Study of the Growth of the of Related Rat Tumours. Brit. J. Cancer, 10:724-32, Walker Rat Tumor and the Flexner-Jobling Rat Car- 1956. cinoma. Am. J. Cancer, 24: 807-22, 1935. 140. WALPOLE,A. L. The Walker Carcinoma 256 in the Screen- 128. SCHULZ, H. Elektronenmikroskopische Untersuchungen ing of Tumour Inhibitors. Brit. J. Pharmacol., 6:135-43, eines Mammakarzlnomes der Ratte. Oncologia (Basel), 1951. 10: 307-29, 1957. 141. WALPOLE, A. L.; WIL~AMS, M. H. C.; and ROBERTS, 129. SELBIE, F. R. Carcinomatous Transformation in a Trans- D. C. The Carcinogenic Action of 4-Aminodiphenyl and plantable Rat Fibroadenoma. Brit. J. Exper. Path., 33: 3:2'-Dimethyl-4-aminodiphenyl. Brit. J. Industr. Med., 61-68, 1942. 9:255-63, 1952. 130. SHAY, H.; AEGERTER, E. A.; GRUENSTEIN, M.; and 142. Wii~o~, R. H.; DEEDS, F.; and Cox, A. J., JR. The KOMAROV, S. A. Development of Adenocarcinoma of the Toxicity and Carcinogenic Activity of 2-Acetamino- Breast in the Wistar Rat following the Gastric Instillation fluorene. Cancer Research, 1: 595-608, 1941. of Methylcholanthrene. J. Nat. Cancer Inst,, 10:255-66, 143. The Carcinogenic Activity of 2-Acetamino- 1949. fluorene. II. Effect of Concentration and of Duration of 131. SHAY, H.; GRUE~STEIN, M.; and HARRm, C. Reproduci- Exposure. Ibid., 7:444-49, 1947. bility of Method and Hormonal Influences in Induction of 144. WRIGHT, A. W., KLINCK, G. H., Jm; and WOLFE, J. M. Breast Cancer in Rats by Gastric Instillation of Methyl- The Pathology and Pathogenesis of Mammary Tumors cholanthrene. Proc. Am. Assoc. Cancer Research, 2:146, Occurring Spontaneously in the Albany Strain of Rats. 1956. Am. J. Path., 16:817-34, 1940. 132. --~. Relationship of Dose of Intragastrically Adminis- 145. ZmBE~, L. A. The Nature of Cancer (Russian Text) tered Methylcholanthrene to Incidence of Breast Cancer Priroda (Mosc.), 4:51-60, 1955: "Abstr.," Excerpta in Rats. Ibid., 3:63, 1959. Medica, 4:183, 1956.

R. L. Noble and J. H. Cutts

Cancer Res 1959;19:1125-1139.

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