Development, Morphology, and Progression of Mammary Tumors During and After Fertile Life in BALB/cfRll1 Mice 1. 2

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1 Development, Morphology, and Progression of Mammary Tumors During and After Fertile Life in BALB/cfRll1 Mice 1. 2 Francesco Squartlnl,3 Marla Blstocchl, 3,4 and Luciano Buongiorno 3,5 ABSTRACT -A study was done on 368 BALB/cfRIIi mammary tumor-bearing breeding female mice that are of the BALB/c genotype and carry milk-transmitted Rill murine mammary tumor virus (MuMTV) infection initiated by foster-nursing. All tumors were characterized by chronologic, biologic, and morphologic analyses. Mammary tumors occurring during pregnancy-lactation periods were mostly pregnancy-dependent plaques showing organoid tubular structure, sudden growth during late pregnancy, regression after delivery, and slow progression. The frequency of pregnancy-dependent tumors per 1 mice at risk increased steadily from first (1 tumor) to sixth (38 tumors) pregnancy-lactation period. Mammary tumors occurring after fertile life were mostly characterized by an irregular, slow growth and a type B morphology. The frequency of these tumors per 1 mice at risk per 1 days of survival also increased regularly in females undergoing from one (73 tumors) to six (117 tumors) pregnancylactation periods. Data were discussed in the light of the pattern of MuMTV release through BALB/ctAlil milk.-jnci 1981; 66: The mechanisms by which milk-transmitted MuMTV induces early tumor transformation of the mammary gland cells are still unknown. Pregnancy-lactation periods increase the incidence of mammary tumors induced by MuMTV in mice, whereas they protect humans against the development of breast cancer (1, 2). In the mouse during pregnancy and lactation, the MuMTV replication within the mammary glands peaks (3). In addition, the amount of MuMTV released in milk increases with each succeeding pregnancy-lactation period (4-6). Thus tumor transformation of the mammary gland cells may be directly related to the amount of MuMTV replicated by these cells. The BALB/dRIII mouse strain, which is of the BALB/c genotype and carries milk-transmitted RIll MuMTV infection initiated by foster-nursing (7), shows the following peculiarities: a) low mammary tumor incidence in virgins (8), b) high production of MuMTV during lactation periods (6, 9), c) high mammary tumor incidence in breeders (1), and d) a large proportion of pregnancy-dependent "plaques" (11, 12), i.e., plaque-shaped mammary tumors growing during pregnancies and regressing thereafter (13-16). Plaques are true tumors because they attain palpable sizes, progress with time toward autonomy, and behave as malignant tumors (17, 18). These peculiarities suggest that in BALB/dRIII mice a sustained replication of MuMTV and hormone stimuli occurring during pregnancy-lactation periods are both required to induce a high incidence of mammary tumors. The question arises as to whether the amount of MuMTV replicated relates to the number of tumors developing in the mammary glands. The alms of this paper are to provide complete chronologic, biologic, and morphologic analyses of mammary tumor development and progression during and after fertile life in BALB/dRIII breeding female mice, to characterize more fully RIll MuMTV, and to elucidate the origins, behavior, morphologic structure, and progression of mammary tumors induced by Rill MuMTV. We shall then discuss the results in the light of quantitative data, previously reported in (6, 9), concerning MuMTV release at lactation periods in BALB/ drill females. MATERIALS AND METHODS We studied 368 BALB/dRIII mammary tumor-bearing breeding female mice that underwent 1,614 pregnancies and developed 831 mammary tumors, 378 of which occurred during pregnancy-lactation periods and 453 of which occurred after fertile life. The average number of tumors per mouse was 2.3. The BALB/dRIII strain was established in 196 from a BALB/c litter transferred immediately after birth to an RIll mother for suckling. Detailed data were reported in (7) concerning the establishment of this strain as well as the mammary tumor incidences in the BALB/c strain of origin, in the milk donor strain RIll, and in the derived strain BALB/dRIII (from Fo to F3o). The pregnancy-lactation period starts at conception of a litter and ends at weaning of the litter; it includes pregnancy (21 days), delivery (l day), and lactation (28 days) and, therefore, lasts 5 days. Female mice were kept continuously with males, 2-3 animals per cage, fed pellets, and given water ad libitum. The reproductive history of each mouse and the occurrence of all palpable mammary tumors were recorded. Each tumor was measured with calipers once a week at the two largest diameters. The mice either ABBREVIATION USED: MuMTV=murine mammary tumor virus(es). 1 Received February 28, 198; accepted August 2, Supported by contracts and from the National Research Council of Italy, Progetto Finalizzato CNR, "Virus." 3 Institute of Pathological Anatomy and Histology, Laboratory of Experimental Oncology, Medical School, University of Pisa, Via Roma 57, 561 Pisa, Italy. 4 Applied Biochemistry, School of Pharmacy, University of Pisa, Via Bonanno 6, 561 Pisa, Italy. 5 We thank Mr. Roberto Marsili for technical assistance and Mrs. Letizia Bardelli for typing the manuscript. 311

2 312 Squartlnl, Blstocchl, and Buonglorno were allowed to die spontaneously or were killed shortly before their natural deaths. At autopsy, tumors were removed for histologic examination RESULTS Occurrence of Mammary Tumors During and After Fertile Life Table 1 gives a complete survey of the mammary tumors observed in BALB/cfRIII mice during and after fertile life, the pregnancy-lactation period, and the tumor type. Text-figure I indicates the total number of mammary tumors detected during pregnancy-lactation periods and after fertile life; the period after fertile life is the time in days elapsed from the last delivery. Most mammary tumors in BALB/cfRIII mice occurred during fertile life or shortly thereafter. A total of 831 mammary tumors were observed in 368 breeders: 378 tumors (45.5%) occurred during pregnancy-lactation periods, 225 (27.1 %) occurred within the next 1 days, and the remaining 228 (27.4%) occurred from 11 to 6 days after the last weaning of a litter. Distribution of Mammary Tumors Detected During Pregnancy-Lactation Periods Text-figure 2 shows the close relationship between mammary tumor development and delivery. The mammary tumor distribution was remarkably uneven because most mammary tumors (246 or 65.1%) were detected during the week before or the week after delivery, whereas only 71 mammary tumors (18.8%) occurred during the first 2 weeks of pregnancy and (/) : 3 ::... >- 25 : "'... ld.. Q. 15 "- 1.; Z DAYS FROM DELIVERY I DELIVERY, DAY I TEXT-FIGURE i.-occurrence of 831 palpable mammary tumors in 368 BALB/cfRIII breeding female mice. Tumors occurring during pregnancy-lactation periods are grouped together. Tumors occurring after fertile life are reported according to the time in days elapsed from last delivery of a litter. only 61 (16.1 %) occurred during the last 3 weeks of lactation. Moreover, most tumors occurring in the 2 weeks around delivery were detected a few days before or after parturition and on the same day of delivery. This observation indicates that mammary tumors appearing during pregnancy-lactation periods were concentrated in excess around delivery, whereas tumor occurrence during early pregnancy or late lactation did TABLE l.-pregnancy-dependent and pregnancy-independent mammary tumors observed d,uring and after fertile life in 368 BALB/cfRIII female mice undergoing 1,614 pregnancies Mammary tumors occurring during pregnancy-lactation periods Mammary tumors occurring after fertile life Preg- Pregnancy-dependent tumors Total nancy- No. of No. of tumors removed for Preg- pregnancylacta- No. of tumors followed No. of dependent Average tion mice by measurements histologic examination nancymice inde- and survival, periods at pendent at days risk Totally Partially Pure Pro- pregnancyrisk gressing Total tumors independent dependent dependent plaques plaques tumors No. of tumors Total 1, After last delivery. ]NCI, VOL. 66, NO.2, FEBRUARY 1981

3 Mammary Tumors In BALB/cfRIII Mice 313 rjj gs 15 : =>... > : : 1 : w -' '" «75 - -'.. "- 5 u..; z 25 DAYS FROM DELIVERY (DELIVERY, DAY 1 TEXT FlGURE 2.-Distribution of palpable mammary tumors oc curring in BALB/C RIII breeding females during pregnancylactation periods according to the day of appearance. Reference is made to delivery (delivery=day ; -days = pregnancy; +days== lactation). not exceed the expected tumor occurrence during nonfertile periods. Characteristics of Mammary Tumors Observed During Pregnancy-Lactation Periods Of the 378 mammary tumors occurring in BALB/ drill breeding females during pregnancy-lactation periods, 246 (6S.1%) were pregnancy-dependent (table 1). This figure was obtained by the sum of tumors followed by measurements that showed total or partial pregnancy dependence and tumors removed for histologic examination around delivery, which revealed the presence of pure or progressing plaques. These tumors appeared early because most of them (189 or 77%) were palpated within the sixth delivery, which occurred at an average age of 327 days (table I). Pregnancy-dependent tumors grew during late pregnancy and regressed a few days after delivery, resumed growth at the successive pregnancy, then regressed again, and so on (text-fig. 3: I-IS). Occasionally, a pregnancy-dependent tumor may have failed to develop at a later pregnancy or a peak of tumor growth and regression may have occurred out of pregnancies, probably as a result of miscarriages (text-fig. 3: 7-8). Pregnancy dependence may be total (text-fig. 3: 1-8) or partial (text-fig. 3: 9-1S). Totally dependent tumors had a plaque shape (figs. 1-4) and an organoid structure made up of peripherally radiated branching ductules (figs. S, 7). Examples of plaque-shaped mammary tumors in BALBI drill breeding females are also given in figures In other papers (17, 18) these tumors were designated as type P (i.e., plaque-shaped) adenocarcinomas. During tumor regression the connective tissue overcame the epithelial structures, and plaques were replaced by a small scar of sclerotic stroma (figs. 6, 12, 13). During the next pregnancy, plaques resumed their growth until they modified the growth curve by progression (text-fig. 3: IS-19). When progression occurred in plaques, it may have been focal or diffuse. Some plaque-shaped tumors (figs. 8-11) may be examples of diffuse progression. Focal progression is more easily recognized, and it usually appears as a round or irregular area of carcinoma within the plaque (figs. 4, 14, IS). After delivery, the remnant plaque regressed but not the area of progression, so that the clinical course was changed, depending then on the growth potential and level of cell differentiation of the progressing focus (text-fig. 3: 9-19). Partially dependent tumors, which may have been stationary or slow-growing (text-fig. 3: 11-14), were either tumors in progression having a slow intrinsic growth rate during which remnant plaques occurred during pregnancy or well-differentiated tu- :13 U a: 2 w I- W :.., o l L L L P W L k L L P <Dill 1lLL L L L L lli tttt L L L L i L L i b!! 4!, I ''a I ;2 TIME IN weeks TEXT FlGURE 3.-Representative sample of growth curves of 21 mammary tumors developed during fertile life in BALB/C RIII breeding female mice. L at the top of vertical lines means litter at the time indicated. P at the top of dotted lines means pregnant at death. 1-8: Totally pregnancy-dependent tumors or plaques. 9-14: Partially pregnancy-dependent tumors : Pregnancy-dependent tumors with subsequent progression. 2-21: Pregnancy-independent tumors. L P,

4 314 Squartlnl, Blstocchl, and Buonglorno mors undergoing milky secretion during the pregnancy-lactation periods. In the absence of these conditions, the tumors originated in plaques by progression behaved independently and grew steadily until the mouse died (text-fig. 3: 16, 18). Almost every plaque during its clinical course in BALB/cfRIII breeding females underwent gradual or abrupt progression toward autonomy (text-fig. 3: 15-19). Pregnancy-independent mammary tumors observed during pregnancy-lactation periods (132 or 34.9%) were indistinguishable in behavior or morphologic characteristics from mammary tumors occurring after fertile life. The curve of pregnancy-independent tumors may be regular (text-fig. 3: 2-21) or irregular. The growth rate also varied. In general irregular curves and slow growth were prevalent. These tumors occurred later in respect to pregnancy-dependent tumors and were concentrated from the fourth pregnancy-lactation period onward. Frequency of Pregnancy-Dependent and Pregnancy Independent Mammary Tumors Occurring at Each Pregnancy-Lactation Period The frequency of pregnancy-dependent mammary tumors, as defined in table I, per 1 mice at risk increased steadily from the first to the sixth pregnancylactation period (text-fig. 4). The tumor frequency was 1.4 at the first pregnancy-lactation period and 37.8 at the sixth pregnancy-lactation period-an increase of 27 times. Each recurring plaque was obviously considered only once: at the pregnancy-lactation period when it appeared the first time. The increase was therefore due to newly arising lesions. The values found approximately tripled at each pregnancy until the third pregnancylactation period, and then they followed a slower progression. After the sixth pregnancy-lactation period, the tumor figures reached a steady state: 39.4 at the <n '" 4 ' >-- <{ 35 "' 3 ""' Q 25 <n " 2 => >-- > cr 15 <{ <{ 1 "' m <i: <{ Q PREGNANCV- L AGTATION PERIODS TEXT,'FIGURE 4.-Frequency (per 1 mice at risk) of pregnancydependent mammary tumors occurring in BALB/cfRIII breeding females from the first to the sixth pregnancy-lactation period. "" (/) cr «"' 11 " S' 1 cr "' "- (/) '" 9 => >-- > «'" 8 '" "' m <I: '" "- 7 PREGNANCY-LACTATION PERIODS TEXT,FIGURE S.-Frequency (per 1 mice at risk per 1 days of survival) of palpable mammary tumors occurring after fertile life in BALB/cfRIII breeding female mice undergoing from one to six pregnancy-lactation periods. seventh pregnancy, 4.5 at the eighth, and 42.8 at the ninth-an increase of 3 times with respect to the first pregnancy (table I). Pregnancy-independent tumors increased also from the first pregnancy-lactation period (.5 tumors/ioo mice at risk) to the sixth (2.7), seventh (26.7), eighth (43.2), and ninth (64.3) pregnancy-lactation periods (table I). The increase was slower during the first pregnancies and faster at later pregnancies. Characteristics of Mammary Tumors Observed After Fertile Life A total of 453 mammary tumors were detected after fertile life in BALB/cfRIII breeding female mice having from to 12 litters (table I). In these tumors various behaviors and histologic types were observed, but a clear predilection was seen for one peculiar pattern characterized by an irregular growth curve, slow growth rate, and type B morphology (19, 2). Mammary tumors showing this behavior totaled 31 or 66.4%. Type B mammary tumors have a "varied" structure with prominent cysts containing secretion, blood, or intracystic papillary projections, and intermingled tubular areas, solid cords, sheets, or nests of cells showing no signs of glandular differentiation (figs ). Mammary tumors occurring after fertile life appeared late. The average ages of the mice when they developed the tumors ranged from 356 days in mice delivering a single litter to 534 days in mice delivering 8 litters. Frequency of Mammary Tumors Occurring After Each Pregnancy-Lactation Period The frequency of mammary tumors detected after fertile life in females undergoing from one to six pregnancy-lactation periods per 1 mice at risk per 1 days of survival is illustrated in text-figure 5. The JNCI. VOL. 66, NO, 2, FEBRUARY 1981

5 average survival in days after the last weaning is given for each group of mice in table 1. The curve in textfigure 5 shows a rather regular increase in mammary tumors, from 72.7 tumors after the first pregnancy-lactation period to tumors after the sixth. The figure after the seventh pregnancy-lactation period was The successive figures were unreliable because of the small number of mice at risk and short survival (table 1). DISCUSSION In the BALBI drill strain, two types of mammary tumors may be detected: a) early appearing tumors, which occur in the mice usually before a year of age and begin as pregnancy-dependent plaques and b) lateappearing tumors, which occur in the mice usually after a year of age and begin as independent tumors, i.e., after the pregnancy-lactation periods. Similar to late-appearing tumors are those that occur during pregnancy-lactation periods (usually late pregnancies) and behave like pregnancy-independent tumors. Pregnancy-dependent tumors originate as duct-like plaques and later undergo focal or diffuse progression to type B or type P adenocarcinomas. The origin of pregnancyindependent tumors and tumors occurring after fertile life is unknown. Probably, these tumors start as hyperplastic alveolar nodules or plaques of submacroscopic size and then progress to type B, slow-growing adenocarcinomas. Both types of tumors are related to milk-transmitted RIll MuMTV (7). Whether RIll MuMTV exists in a single form or in two forms transmitted together to offspring and each is responsible for one type of tumor are matters of speculation. In some sublines of the RIll strain from the United States, the resident MuMTV has lost its ability to induce plaques (Moore DH: Personal communication) but is still able to cause pregnancyindependent tumors. Quantitative relationships between milk release of RIll MuMTV and occurrence of mammary tumors in BALB/dRIII females deserve further consideration. In BALBI drill breeding females, a high production of MuMTV takes place during lactation periods, though with appreciable individual variations (6). By plotting these individual variations against the age of the animal at appearance of the first palpable mammary tumor, investigators have previously observed a direct relationship between MuMTV replication and tumor development. The higher the amount of MuMTV released, the lower was the age of the mouse at onset of mammary tumors (9). The BALB/dRIII strain exhibits a low mammary tumor incidence in virgin females and a high mammary tumor incidence only in breeding females (8, 1). Two possible explanations of this fact are: '1) A sustained replication of RIll MuMTV, which occurs only at pregnancy-lactation periods, is required to induce mammary tumors and 2) RIll MuMTV is oncogenic only when associated with the hormonal Mammary Tumors In BALB/cfRIII Mice 315 stimuli of pregnancy-lactation periods. These possibilities are in line with the observation that many mammary tumors of BALB/dRIII breeding females begin as either pregnancy-dependent plaques or plaques in progression. Plaques have so far been observed only during pregnancy-lactation periods (13-16) and only in strains of mice carrying plaque-forming variants of MuMTV; examples of such strains are Rill (21-23), GR (17, 18, 24), DD (25-27), and DDD (28). Pure plaques are organoid structures starting from a center and made up of peripherally radiated branching tubules. They appear quickly during late pregnancy and later on disappear quickly. In this respect, plaques may be assumed to be an expression of a phenomenon of quickly appearing, focally detectable, tumor cell transformation by RIll (or other similar) MuMTV. Plaques occur during periods of high MuMTV replication associated with sustained and peculiar hormone stimulation. The threshold for their occurrence at pregnancy-lactation periods might therefore depend on the rate of MuMTV replication. In fact, a direct relationship has been found between the frequency of pregnancy-dependent palpable mammary tumors detected and the amount of MuMTV replicated at each pregnancy-lactation period in BALB/dRIII females; both these figures increase steadily and progressively from the first pregnancy-lactation period to the sixth and successive pregnancy-lactation periods. Release of MuMTV in milk at the sixth lactation is approximately seven times that at the first lactation (9). The frequency of pregnancy-dependent mammary tumors occurring during the sixth pregnancy-lactation period is approximately 27 times that observed during the first pregnancy-lactation period; the figure rises again up to 3 times during the ninth pregnancy-lactation period. However, MuMTV alone is unable to maintain pure plaques because these regress during lactation when a high replication of MuMTV also occurs. Only the association of MuMTV and the hormones of pregnancy or sc transplanted hypophyses (29, 3) can maintain plaque growth. This may be explained if one assumes a type of primarily hormone-dependent tumor cell transformation by RIll MuMTV, which, as it happens, becomes independent later on. Pregnancy-independent mammary tumors occurring during fertile cycles and the mammary tumors that develop after fertile life in BALB/dRIII breeding females also increase in frequency with the number of pregnancy-lactation periods. Therefore, the frequency of these tumors also shows a direct relationship with the amount of MuMTV replicated by the host mice before tumor appearance. In this respect, the favorable influence of pregnancies on mammary tumorigenesis in mice should be reconsidered to ascertain whether the oncogenic role of hormones released during pregnancylactation periods depends on a) sustained replication of MuMTV which the hormones cause, b) promoting action, or c) tumor transformation of virus-infected cells. Data have previously been reported that show direct

6 316 Squartlnl, Bistocchl, and Buonglorno quantitative relationships in strains having a high incidence of mammary cancer between MuMTV production during lactation periods and mammary tumorigenesis in the mammary glands (4, 5, 9, 31-33). Such data might contribute to an understanding of the mechanisms of mammary gland cell transformation by viruses. The data presented here provide a further approach to this understanding. REFERENCES (1) SEVERI L, SQUARTINI F. Problems arising in the control of breast cancer. Acta VICC 1962; 18: (2) MACMAHON B, CoLE P, BROWN J. Etiology of human breast cancer: A review. J Nat! Cancer Inst 1973; 5: (3) LASFARGUES EY, FELDMAN DG. Hormonal and physiological background in the production of B particles by the mouse mammary epithelium in organ cultures. Cancer Res 1963; 23: (4) CHARNEY J, PuLLlNGER BD, MOORE DH. Development of an infectivity assay for mouse mammary-tumor virus. J Nat! Cancer Inst 1969; 43: (5) VERSTRAETEN AA, VAN NIE R, KWA HG, HAGEMAN PC. Quantitative estimation of mouse mammary tumor virus (MTV) antigens by radioimmunoassay. Int J Cancer 1975; 15: (6) BISTOCCHI M, NUTI M, SQUARTINI F. Quantitative comparison of milk-released C3H and Rill mammary tumor viruses in infected BALB/c hosts. Tumori 1977; 63: (7) SQUARTINI F, BoLlS GB, ROSSI G. BALB/cfRIII: A new high mammary-tumor and high leukemia mouse strain. J Nat! Cancer Inst 1974; 52: (8) SQUARTINI F, BISTOCCHI M. Bioactivity of C3H and Rill mammary tumor viruses in virgin female BALB/c mice. J Nat! Cancer Inst 1977; 58: (9) SQUARTINI F, BISTOCCHI M, PASINI G. The maternal milk route in the causation of early murine tumours: Relationship between amount of MTV released and mammary tumorigenesis in BALB/cfRlI1 mice. In: Severi L, ed. Tumours of early life in man and animals. Perugia: Division of Cancer Research, 1978: (1) SQUARTINI F. Multiple cancer in the mammary gland system: A murine model suitable for human beings. In: Severi L, ed. Multiple primary malignant tumours. Perugia: Division of Cancer Research, 1975: (11) SQUARTINI F, ROSSI G, PAOLETTI I. Characters of mammary tumours in BALB/c female mice foster-nursed by C3H and Rill mothers. Nature 1963; 197: (12) SQUARTINI F. Relationship between mammary tumor virus and other oncogenic viruses in mouse mammary tumorigenesis. In: Carcinogenesis: A broad critique. Baltimore: Williams &: Wilkins, 1967: (13) FOULDS L. Mammary tumours in hybrid mice: Growth and progression of spontaneous tumours. Br J Cancer 1949; 3: (14) ---. The histologic analysis of mammary tumors of mice. II. The histology of responsiveness and progression. The origins of tumors. J Nat! Cancer Inst 1956; 17: (15) SQUARTINI F. Responsiveness and progression of mammary tumors in high-cancer-strain mice. J Nat! Cancer Inst 1962; 28: (16) SQUARTINI F, ROSSI G. Responsiveness and progr.ession of the morphological precursors of breast cancer in inbred mice: A review. In: Severi L, ed. The morphological precursors of cancer. Perugia: Division of Cancer Research, 1962: (17) VAN NIE R. Behaviour and morphology of pregnancy responsive mammary tumours in mice. Pathol Eur 1967; 2: (18) VAN NIE R, Dux A. Biological and morphological characteristics of mammary tumors in GR mice. J Nat! Cancer Inst 1971; 46: (19) DUNN TB. Morphology of mammary tumors in mice. In: Homburger F, ed. The physiopathology of cancer. 2d ed. New York: Harper, 1958: (2) SQUARTINI F. Tumours of the mammary gland in the mouse. In: Turusov VS, ed. Pathology of tumours in laboratory animals. Vol II. Tumours of the mouse. Lyon: IARC, 198:43-9. (21) SQUARTINI F, ROSSI G. Accrescimento e progressione dei tumori mammari nei topi femmina del substrain RIII/Dm/Se. Lav 1st Anat Istol Patol Perugia 1959; 19: (22) ---. Analisi morfologica della "responsiveness" e della progressione nei tumori mammari del "substrain" RIII/Dm/Se. Lav 1st Anat Istol Patol Perugia 1959; 19: (23) SQUARTINI F, SEVERI L. 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7 Mammary Tumors In BALB/ctRIll Mice 317 s FIGURES Tumor sections from BALB/cfRIII female. x 12.5 FIGURE I.-At the end of si:kth pregnancy. Left abdominal breast. S=skin; P=plaque; MG=mammary gland. Bar= I mm. X FIGURE 2.-Day after sixth delivery. Right thoracic breast. Plaque. Bar= I mm. X FIGURE 3.-Day after fifth delivery. Left adbominal breast. Plaque. Bar= I mm. X FIGURE 4.-In the course of third pregnancy. Left thoracic breast. Plaque with area of focal progression (T). Bar= 1 mm. X FIGURE 5.-Day of third delivery. Right thoracic breast. Incomplete plaque. Bar= 1 mm. X FIGURE 6.-Twentyfive days after fifth delivery. Right thoracic breast. Regressed plaque. Bar= I mm. X FIGURE 7.-At the end of sixth pregnancy. Right abdominal breast. Edge of plaque: Note the peripherally radiated branching tubules. Bar= I mm. X 3

8 318 Squartlnl, Blltocchl, and Buonglorno FIGURE 8.-Three days after fourth delivery. Right inguinal breast. Plaque-shaped tumor (T) in "mamma lactans" (ML). Bar = I mm. X FIGURE 9.-Twenty-four days after sixth delivery. Left thoracic breast. Plaque-shaped tumor in diffuse progression. Bar = 1 mm. X FIGURE 1.-In the course of fourth pregnancy. Left inguinal breast. Plaque-shaped tumor. Bar = 1 mm. X FIGURE 11.-Three days after fourth delivery. Left thoracic breast. Plaque-shaped tumor. Bar = 1 mm. X FIGURE 12.-Thirty-seven days after sixth delivery. Right abdominal breast. Regressed plaque. Bar= 1 mm. X FIGURE 13.-ne hundred fifty-two days after seventh delivery. Left thoracic breast. Regressed plaque. Bar = 1 mm. X 12.5

9 Mammary Tumors In BALB/cfRIll Mice FIGURE 14.-The day after sixth delivery. Right abdominal breast. Plaque (P) in focal progression (T). Bar= I mm. X FIGURE 15. Seven days after miscarriage. Right inguinal breast. Focal progression (T) in plaque (P). Bar= I mm. X FIGURE 16.-Forty-nine days after fifth delivery. Right abdominal breast. Adenocarcinoma type B. Bar = 1 mm. X FIGURE 17.-Seven days after miscarriage. Right cervical breast. Adenocarcinoma type B: Detail. Bar=1 mm. X 22. FIGURE IS.-One hundred eighteen days after fifth delivery. Left abdominal breast. Adenocarcinoma type B: Detail. Bar = I mm. X 22