A COMPARATIVE STUDY OF THE LIFE HISTORY OF THE FEMALE IN TWO STRAINS OF ALBINO MICE * LYDIA M. GIBSON (FTo~the Department of Pathology, Yale University School of Medicine) In searching the literature nothing could be found concerning the life history of the mammary gland in mice, although much work has been done on other rodents. Since the mouse is used so extensively in cancer research as an experimental animal, and since cancer of the mammary gland is a well recognized entity in certain strains of mice, it was thought that it would be interesting to make a comparative study. To do this, two strains of mice were necessary, one showing a low incidence of mammary cancer in the females, and the other showing a high incidence. The material presented here represents an attempt to work out the development and distribution of mammary tissue in two such strains of mice. The study is a preliminary one, and the material is limited, but it was decided that should the two strains exhibit any remarkable differences, a more extensive survey might prove both interesting and profitable. The high tumor incident strain has been designated through- out as SPTM, and the low tumor incident strain as Loomis 1. Bagg strain was abandoned because of lack of material. Data concerning the animals used in this study are found pre- ceding the plan of work. PURPOSE. By gross and microscopic study (1) to trace the development and distribution of mammary tissue in female mice; (2) to compare two strains: (a) one having a low mammary car- cinoma incidence (no strain that has been studied carefully has been known to be tumor free) and (b) a high spontaneous mam- * A thesis submitted to the Faculty of the Yale University School of Medicine in candidacy for the degree of Doctor of Medicine. 570 mary tumor strain; (3) to find, if possible, when deviations from the normal begin to occur, and to trace these deviations to the developed neoplasm. MATERIAL:The material for this work is drawn from the fol- lowing sources : I. Control Strains: (1) Bagg (low mammary tumor). Re- ceived one pair of breeders March 20, 1928. Albinos from stock of albino strain established 1913. Mice received from dealer in Ohio, inbred by brother and sister mating, no new blood added; breeders selected for vigor and breeding qualities; no other infor- mation available except that these mice have shown a low in- cidence of mammary carcinoma. (This refers to female mice). (2) Loomis 1. Four males and ten females received April 12, 1928; ten females and two males received May 25, 1928; three females and one male received April 12, 1928; donated by Dr. Lewis, Phipps Institut,e, Princeton. Original stock from Cam- den, N. J. Sixteenth generation of brother and sister matings (G. stock). No mammary carcinoma ever observed in this strain. II. Spontaneous Mammary Carcinoma Strain: Twenty-one females and two males received March 22, 1928, from the State Institute, Springville, N. Y. Thirteen females and eight males were received on July 23, 1928. Original stock obtained from Abbie Lathrop, Cranby, Mass. No information was secured except that the mice were from high tumor stock. None were identified as high or low representatives or in any other way. They were bred at random by M. C. Marsh at Springville, N. Y. At least nine inbred strains were carried out, before finding the one that is the present high tumor strain (known as strain 3). The mice used in this experiment were offspring of this high tumor strain. Tumors observed in these mice were predomi- nantly mammary carcinoma, and these neoplasms have been observed only in females. This is the twenty-fifth generation; the last few generations have yielded 80 per cent tumors. Sarcomas are rarely observed in these mice. Tumors develop in the fifth to*the nineteenth month. The highest development is reached in a year and a half to two years. No mammary car- cinoma has ever been observed in male mice of this strain. 572 LYDIA M. GIBSON Diet: All mice were fed on an adequate mixed diet, and a sup- plementary feeding of milk powder was given to the nursing mothers. Breeding: Strains have been kept separate and inbreeding carried on. Details of exact data concerning mating, gestation, weaning, and age will be found in the individual records. Plan of Work: Using histological preparations for study: Control Spontaneous Tumor Strain 1. Trace the development of 1. Procedure same as control. the mammary glands from the first indications of the milk line in the embryo to the development of the with the underlying immature gland tissue. This requires sectioning embryos of different stages in their development. 2. Series of sections showing 2. Procedure same as control. the state of glands at birth. 3. State of glands at varying 3. Procedure same as control. age periods (one month, two months-puberty, three months-mature) . 4. Lactating . 4. Procedure same as control. 5. Adult resting breast. 5. Procedure same as control. 6. Series of sections of breast 6. Series of sections showing, tissues from adult females, different types of breeders and non-breeders, and a series similar to the from the active reproduc- control, concerning atrophic tive period to the atrophy of changes. In addition, a ser- old age. ies showing the occurrence and distribution of neo- plastic growths at different age intervals. FEMALE MAMMARY GLAND IN TWO ALBINO MICE 573

PROCEDURE.1. For the embryos, the animals were mated. The copulatory plug read and the date recorded, the animals were killed on the tenth, fourteenth, nineteenth days from the dis- covery of the plug. Embryos were removed from the uterus and fixed in 10 per cent formalin. After imbedding in paraffin, serial sections, 10 micra thick, were made. Several attempts to secure a nineteen-day embryo in the control strain were made, but in one case the uterus was opened and the embryos were partially absorbed, and in three other cases the animals refused to mate; in the fourth case the plug was read, but due to unfore- seen circumstances the animal came to term and delivered. Time shortage prevented further mating, and this embryo had to be omitted. 2. Skip serial sections, 10 micra thick, every tenth section used on all animals sectioned from birth through three-month devel- opment. Animals at birth were fixed in 10 per cent formalin, decapitated to cut down extra expense of sectioning, and cross sections through the whole body were made. From the tenth day to the third month, the following procedure was carried out: For the tenth day post-natal animals, the skin was removed by slitting it up the center of the back, cutting it at the root of the neck and at the base of the tail, and peeling it off glove-fash- ion from the body. The deposits of fat in the inguinal and tho- racic regions came with it. This skin was then spread out on a blotting paper and pinned in position, labelled, and fixed in 10 per cent formalin. Drawings were made of the location of the nipples and the fatty deposits. When the skin was fixed, it was divided by cutting down the mid-line of the ventral surface. One-half was used for sectioning and the other half was saved. The band of tissue between the thorax and the abdomen, which is free from nipples and fat deposit, was discarded. The tenth day post- natal animals were so small that the whole portion could be sec- tioned. But the areas so obtained in the first, second and third month development were too large to section in one piece, so the thoracic and inguinal deposits were divided into three portions and sectioned. Rough drawings were made for identification, e.g. thoracic deposit labelled th. A, B, C and ing. A, B, C. By 37 574 LYDIA M. GIBSON this scheme, which worked out fairly well, the whole deposit, in- cluding the nipples, was sectioned. For the older mice (six months onward), single sections were made from tissues taken either from the thoracic or inguinal deposits, as indicated in the notes.' It was not thought neces- sary to section the older mice so extensively, since this procedure was carried out thoroughly up to maturity, and a fair conception of the distribution was gleaned from the study of these slides. Note: Measurements were made on the embryos from the head end to the tail end. The embryo was laid on a centimeter ruler and its length was ascertained. Measurements Control Spontaneous Tumor Mice 10 day embryo L.I.G. 3 mm. 10 day embryo SPTM 3 mm. 14 day embryo L.I.G. 10.5 mm. 14 day embryo SPTM 11 mm. 19 day embryo L.I.G. 19 day embryo SPTM 15 mm. At birth L.1.C:. 15 mm. At birth Animals were not weighed or measured after this period. Slides were studied carefully and written up. Photographs were made of interesting areas and are included with the data. EMBRYONICDEYELOPMENT: The mouse follows the general plan of mammalian development. The mammary streak and the mammary line doubtless appear shortly after the tenth day of embryonic life. I was unable to identify the line in either of the embryos of this age, but remnants of the line are still recognizable in embryos of fourteen days. This line of heaped up epithelial cells extends from the root of the neck to the groin on each side of the body, lateral to the midline. Along this line the future nipples develop. The number of nipples is characteristic of the species. The mouse has five on either side of the body, three in the thoracic region, and two in the inguinal region. The portion of the line between the thoracic and the inguinal regions disap- pears. Comparison of the two embryos (SPTM and Loomis 1) at ten days of age, showed them both to be in a very elementary state of development, but the SPTM specimen showed an indication FEMALE MAMMARY GLAND IN TWO ALBINO MICE 575 of limb buds and was, therefore, considered to be in advance of the control strain which did not show such development. The first indication of the future nipples is a sinking-in of the ectodermal cells along the milk line. This invagination marks the site of the future . The column of solid epithelial cells that continues to grow downward from the surface repre- sents the anlage of the primary lactiferous which later gives rise to the secondary ducts and alveoli of the true mammary gland. There are no skin glands developed at this stage. In both the embryos of this period (fourteen days) this phe- nomenon is well illustrated. However, this SPTM embryo seems to be more advanced in development of this region. Sex was determined by measuring the distance between the anal pore and the genital papilla. Unfortunately, the nineteen-day embryos cannot be compared. The SPTM embryo will be described, since the control is missing. There is very little change in the picture except that the down- growth of the column of epithelial cells is more definite and a little more pronounced. They are making their way in each instance lateralward toward the embryonic fat deposits which are laid down in the thoracic and inguinal regions. There are five of these columns on either side, three in the thoracic region and two in the inguinal region. The pits left by the invagination of the surface epithelial cells are being filled in by cornified cells. These zones now have the appearance of a marked keratosis when compared with the surface covering adjacent to them. The small central indentation remains and indicates the future opening of the primary milk duct. The fat deposits mentioned above, toward which the columns of cells are working, are not very well defined in either the tho- racic or the inguinal region. In the thoracic region, the area is highly vascular, and the embryonic fat cells are found below the corium wedged in between it and the band of muscle (panni- culus carnosus) lying just below. They are meager over the anterior chest and fan out toward the axillary region, where they are easier to outline. The inguinal region shows an irregular mass of fat cells almost meeting in the midline, concentrating in 576 LYDIA M. GIBSON a fairly well defined mass in each groin. The anlagen of the future lymph nodes are identified about the center of each de- posit. AT BIRTH:We again have two animals to compare. The con- trol animal originally collected was ruined during embedding. Since time did not permit a second mating of a new pair, an animal twenty four hours old was substituted.

Flu. 1. CONTROLL. 1. FOURTEEN-DAYEMBRYO. Seetion through nipple zone. Th. 3 showing (a) invagination of the ectoderm, with (b) ttie ar~lageof the primary milk duct. X 75. The sections were gone over carefully, and it was decided to select the zone at the level of the third thoracic nipple as being representative of the development of this period. In three days there is a marked advancement in the picture. The pit has filled in, the central opening or pore remains, and the nipple has de- veloped so that it is recognizable as such. It has a definite shape, projects a bit above the surface, has a thick epithelial surface covering, and a vascular, well developed stroma. There is a sulcus on either side of the nipple marked off by a dipping-in of the epithelial cells. FEMALE MAMMARY GLAND IN TWO ALBINO MICE 577

The original solid column of cells has thinned out, has acquired a lumen, and has become the primary lactiferous duct. This main duct is seen coursing through the center of the nipple, al- most perpendicular to the skin surface. It is patent and com- municates with t,he exterior through the milk pore; branching of the duct in this region does not seem to occur. The epithelial

Fxo. 2. SPTM. Section througll second inguinal nipple zone, fourteen-day embryo; showing formation of (a) the pit, with a well developed unlage (b) of the pri- mary lactiferous duct. (c) Remnants of the old milk ridge. X 125. cells lining the duct are low cuboidal in type, two rows deep, and are more succulent looking and stain deeper than the surface epidermal layer. They more closely resemble the cells of the basal layer. As the duct passes through the nipple, it is sup- ported by a connective-tissue stroma. It curves gently lateral- ward toward the fatty tissue of this region, to end blindly in this deposit. There are no alveoli formed at this time. The fatty deposit is lobulated and vascular. Both inguinal and thoracic lymph nodes are formed. The inguinal primary ducts in both instances show branching, forking out to end blindly in the inguinal fat deposit. 578 LYDIA M. GIBSON The outstanding differences between the animals from the two strains, at this stage, are as follows: Macroscopically, the control strain showed poorly defined nipples; microscopically, the nipples are seen to be present but to have remained sessile. They are well developed, but are on a level with the skin surface and, although there is a gentle round-

FIG.3. SM'M. NINETEEN-DAYEMBRYO. Section through nipple zone at Th.3 showing (a) pit being filled in with cornified cells, and (b) the anlage of the primary milk duct. X 125. ing of the central portion, the marked projection and definite cone-shape noted in SPTM are absent. The fatty deposits in this animal were lobulated. There were no acini in either of these deposits. Neither animal showed any indication of such development at this period. TENDAY POST-NATAL: Bagg female used here as a control (sectioned before the ~ccident)SPTM-skip serial type, as noted in the procedure. After birth, these two animals of the same age show a parallel development. Therefore, the following description will suffice FEMALE MAMMARY GLAND IN TWO ALBINO MICE 579 for both. The five nipples on either side of the body can easily be located macroscopically. The first three pairs are placed high on the thorax, lateral to the midline, almost in the axillary line. The first nipple is at the root of the neck and nearest the midline, the second nipple about the level of the midaxillary

FIG.4. CONTROLL. 1. TWENTYFOUR HOURS OLD. (a) Nipple formed but sessile; (b) primary duct with patent lumen. X 75. region, and the third midway between the axilla and the inferior costal margin. The fatty deposits are easy to map out. That of the thoracic region, being less compact, spreads loosely from a point below the midclavicular region. Here it is deepest. It extends also for a short distance up on the dorsum, terminating just lateral to the angle of the scapula. On microscopic examination (thoracic region), the nipples are seen to be well developed, with the primary lactiferous ducts patent and opening to the surface through the milk pores. The ducts pass through the nipples of this region and curve lateral- ward toward the fat deposits. The intradermal portion of the duct in each case is almost at right angles to the skin surface. The primary ducts of Th. 1 and Th. 2 are found buried in the 580 LYDIA M. GIBSON upper portion of this deposit and their branches ramify in it. The duct which leads from Th. 3 works toward the axillary region and branches as it reaches this part of the fat deposit. Each nipple has its own primary duct, and each primary duct has it,s

FIG.5. SPTM. At BIRTH. Section through nipple (a) and primary duct (b) Th. 3. (c) Milk pore opening to surface. X 125. own branches. As far as could be made out, each system is en- tirely separate and does not communicate with the other, al- though the branches of the ducts lie in close approximation. The lobular structure of the fat deposit is noticeable. There is no development of alveolar tissue at this stage. There is no lymph node seen, but the blood supply to this area is plentiful. The inguinal region, grossly, shows the two nipples on either side. The fifth pair of nipples (In. 2) are placed very near the midline and just above the genital papilla. The fat deposits of this region are more extensive. They start near the midline on

FIG.6. CONTROLBAOG. TEN-DAY POST NATAL. Nipple (a),primary milk duct (b), fat deposit (c). Duct has made its way into the deposit. (Th.) X 75.

either side of the body, about the level of the fifth nipple; passing lateralward and upward, they arch over each hind leg, toward t,he dorsum. The deposit fits into the curve of the groin; for this reason, it has a pyramidal shape, with its apex pointing downward. A thin veil of fascia covers this deposit, making it easily discernible from the surrounding tissues. The ramifica- tions of the primary from In. 1 and In. 2 are received in this deposit . Microscopically, the nipples of this region are seen to project a little above the surface, but to be placed obliquely, parallel to the hairs, with the tip of the nipple pointing caudad. The sulcus surrounding the posterior portion of the nipple, and the zone 582 LYDIA M. GIBSON immediately above it, are free from hair. The sebaceous glands are well developed and are most plentiful where the hair is thick- est. No sweat glands are present. The intradermal portions of the primary ducts have a shorter course than in the thoracic region, but they run superficially below the corium for a greater distance. They end blindly, by forking out into the inguinal fat pad. There is no attempt as yet to form acini. The lymph

FIG.7. SPTM. TEN-DAYPOST NATAL. Nipple sessile (a), primary duct (b), and fat deposit (c). (Th.)X 75. node is well developed and placed about the center of the deposit. ONE MONTH(RUDIMENTARY) : Loomis 1 : grossly, all nipples are visible; inguinal and thoracic fat deposits are present and well defined. SPTM; animal puny; grossly, nipples are not well defined, but the thoracic and inguinal fat deposits are made out. Owing to the fact that these animals show a marked difference, they will be described separately. The control animal, Loomis 1, shows the presence of nipples which are well formed and well developed. There is a more pronounced branching of the primary ducts of the thoracic and inguinal lactiferous systems. There is also a notable pentration into their respective fat deposits. The lymph nodes of the tho- racic and inguinal regions are found, but there is still no trace of acinar structure at the ends of the ducts or their branches. The SPTM shows grossly a lack of nipples, both thoracic and inguinal. Microscopic examination fails to locate the thoracic group and reveals only a few tiny superficial, atrophic ducts. No trace of the fourth nipple was found, but the fifth one showed a malformation, with an immensely dilated duct (see Fig. 9). It would seem that there was an anomaly in this animal. It has been noted that, occasionally, nipples are missing in this strain, often one or more of the thoracic group. This refers to gross examination, and it was an incidental finding when other ex- perimental work was being done with animals from this strain. Also, it might be added, the nipples on the right side of the body have a tendency to range slightly higher than those on the left. These sections were taken from the right side of the body. Two MONTHS(PUBERTY) : Loomis 1: grossly, well developed, well nourished; nipples visible; fat deposits of both regions are present. SPTM: small puny animal; nipples not well developed and difficult to locate; fat deposits all present, and left inguinal greater than right. The main difference in these two specimens was discovered macroscopically, i.e., the apparent under-development of the nipples in the SPTM. Microscopically, the first and second thoracic nipples were sessile, but both possessed a duct system. .4n epidermoid was noted in the skin of the chest near the second nipple (Th.). Since both animals showed the following points in common, a description of sections through Th. 3 will suffice. These sections are very interesting because they show a marked activity, which is in contrast to those studied with refer- ence to the one-month development. The primary duct with its secondary offshoots ramifies more extensively in the lobulated thoracic fat deposit. The difference between the cells lining the intradermal portion of the duct and those at its terminal portion 584 LYDIA M. GIBSON is for the first time noticeable. The low cuboidal epithelial cells are flattened out almost to a thin line, as the main stem of the primary duct threads toward the nipple. As we progress toward the end of the duct, these lining cells gradually become higher and deeper stained. The terminal part and the secondary branching of this duct are lined with succulent . Distinct knobs of these cells are protruding from the blind ends of the duct and its offshoots, and are budding out into the fat. This is the formation which will produce the acini of the adult mammary tissue. Every lobule of fat has patches of these densely stained cells scattered through it. The branching of the main ducts has been quite active to produce this picture. The inguinal region shows a similar picture. The lobulation of the fat deposits is very noticeable now, as is the network of vessels and nerves that thread through them. Each deposit shows well defined lymph nodes, and the lymphatics of the area apparently drain toward these nodes. The thoracic zone has two and the inguinal has one node centrally placed. In relation to the nerve supply, it appears that the thoracic region has two main plexuses placed just beneath the corium; the inguinal nerves are not so clear cut. The thin band of muscle lying quite super- ficially above the is also characteristic of the thoracic region. THREEMONTHS (MATURITY) : Loomis 1 : grossly, nipples are all present and well developed (Th. and Ing.); fat pads easily demonstrable (Th. pads small). SPTM: grossly, nipples cRn not be made out; Th. and Ing. fat pads equally well defined. Microscopic examination of the sections from these two ani- mals reveals several interesting facts. In the first place, the control animal has well formed nipples. The primary lactifer- ous ducts and milk pores are patent. Branching of the ducts has been abundant, so that they spread diffusely throughout the fat deposits in their respective localities. There is alveolar formation at the ends of the secondary offshoots. Other sections show masses of densely packed cells at the ends of the twigs as they jut out into the adipose tissue. The racemose structure of the gland is emphasized by the lobulation of the fat. Every branch of the main trunk is encased in a lobule of fat. The budding alveoli push into it, carrying with them a fine connective-tissue stroma which serves as a basement membrane for the epithelial lining cells. The main duct, as has been de- scribed previously, is supported in its course by a firm connective stroma upon which the double row, cuboidal epithelial cells rest.

FIG.8. CONTROL.ONE MONTH DEVELOPMENT.Thoracic fat deposit (a); prininry duct (b); lymph node (c). A few secondary branches of the duct are seen, cross section. X 75. The vessels and nerves with the lymphatics are held in position by this same means. There is evidence of secretion in the ducts; a homogeneous substance fills the lumina. Lymph nodes are found in both regions. A dissimilarity between the two animals certainly exists. The SPTM shows unerupted nipples; they have all formed but lie sunk in the stroma of the corium. The lactiferous ducts are not unlike those in the control strain, but the alveolar structure is developed to a far less extent and is not so exuberant. Secre- tion is also noted in the ducts. The architecture of the sebaceous 586 LYDIA M. GIBSON glands is distorted, and the skin shows a marked polymor- phonuclear reaction in many places, as a result of the irritating effect produced by infesting mites.

FIG.9. SPTM. ONEMONTH. (a) Malformed second inguinal nipple; (b) dilated duct. LACTATINGBREABT: One description will suffice for both strains, since there is no remarkable difference between them. Grossly, the fat deposits of the inguinal and thoracic regions have lost their compact, opaque appearance, and have become milky white. The veil of fascia covering them is so transparent that the detail of the fretwork of the gland can be made out on close inspection. They have hypertrophied to such a degree that they now cover three times the zone they formerly occupied. The cut surface is spongy and oozes milk. Microscopic examination shows a lace-like tissue extending from beneath the true derma down to the edge of the fat de- posit of the region. Closer inspection suggests that the small

- FIG.10. CONTROLL. 1. TWOMONTHS DEVELOPMENT. THORACIC REGION. Showing (a) marked branching of the ducts in the fat deposit, and (b) the begin- ning alveolar buds. X 75. compact alveoli of the virgin gland have suddenly ballooned out and have multiplied to an enormous extent. The individual alveoli, widely distended with secretion, have expanded to an immense size, and are seen cut at all angles. The ducts and their branches are also hypertrophied, and, on cross section, it is difficult to make out in some instances which is duct and which is alveolus. But the walls of the alveoli are thinner and they are lined with a single row of cells. The terminal portions of the secondary ducts show the expansion into acini very nicely. The lining cells of the alveoli are in all stages of activity. Some cells are large and vesicular, with pale staining nuclei; other 588 LYDIA M. GIBSON have a fluffy protoplasm; others appear swollen to the bursting point. In many cases the cell wall has ruptured, and a globule of fatty substance is being extruded into the lumen. In such cells the nucleus is flattened against the connective-tissue frame- work.

Fra. 11. SPTM. Two MONTHSDEVELOPMENT. Th. 2. (a) Nipple sunk in stroma of corium; (b) primary duct; (c) branch of duct. Notice lobulation of fat de- posit and paucity of branching and alveolar budding. (d) Alveolar bud. X 75. Many of the acini are quite free from secretion; in these the lining cells have ti wrinkled outline. A certain number are gorged with secretion, and their lining cells are pushed back against the basement membrane by the material held within. The original lobulation of the fatty deposit remains; but the fat is almost entirely replaced by hypertrophied gland substance. There is a fine network of supporting connective tissue running through the deposit, which carries the blood vessels, lymphatics, and nerves.

FIG.12. CONTROLL. 1. THREEMONTHS DEVELOPMENT. DUCTB AND ACINIIN FATDEPOBIT. THORACIC REGION. (a)Duct with secretion in lumen; (b) secondary branching; (c) alveolus. X 75.

The nipples are also hypertrophied, projecting considerably above the surface. The primary ducts are distended, as are all portions of the collecting system. Secretion is plentiful in their lumina. Lymph nodes of each region show a slight enlargement. ADULTRESTING: Tissues from both strains of animals show a picture that corresponds to the three-month development, ex- cept that there is a greater amount of connective-tissue stroma throughout the gland. There is a small amount of brownish yellow pigment noted in the deposits of mice six months old. To digress here, one SPTM (six months) shows beautifully the 38 590 LYDIA M. GIBSON mammary gland involuting after . The whole mass of hyportrophied alveolar tissue seems to be receding, and the adipose tissue again comes into prominence. The islands of acini still are thickly distributed in the fat lobules, and the spongy appearance of the cells, with dilatation of the ducts and vessels in their immediate vicinity, is evidence of recent activity.

FIG. 13. SPTM. THREE MONTHSDEVELOPMENT. DUCTS AND BRANCHES. THORACICREGION, WITH A FEWALVEOLI. (a) Alveolus; (b) primary duct with aecre- tion in lumen; (c) secondary branch of duct; (d) fat deposit. X 75. The nipples are markedly larger than those of the virgin mouse and project well above the surface. One of the control animals in this age period showed multiple abscess formation throughout the whole body. In the mam- mary regions there was a typical exudative polymorphonuclear reaction, with the formation of many small abscesses in the de- posits. Other control animals are more than six months old and demonstrate the atrophic changes that accompany age; these will be described subsequently. ATROPHYOF OLD AGE AND MALIGNANTNEOPLASMS: (SPTM stock, six months to twenty-two months, inclusive. L. 1 stock, five and a half months to seventeen months, inclusive): As age advances, both strains of animals show the following: There is loss of hair, especially over the thorax and belly. This brings all the nipples into prominence; the fat pads of both

FIG. 14. SPTM. LACTATINGBREAST. Section through thoracic deposit, showing lobulation of the gland; (a) distended acini; (b) duct; (c) lymph node. regions become firmer, and pigmentation is marked. Many of the old SPTM showed excoriations on the chest wall and cystic degeneration in the deposits, as well as tumor formation. The control strain lacks any indication, grossly, of such changes. If we assume that the L. 1 stock (control) is representative of 592 LYDIA M. GIBSON

.I the normal, then we should expect that the changes observed in these mice are characteristic and concomitant with the advance of age. The SPTM show a parallelism up to a certain point, and

FIG.15. CONTROL.AGE SIXTEENMONTHS. ATROPHICCHANGES. TH. DE- POSIT. (a) Primary duct with increase in connective tissue surrounding it and nar- rowing of lumen; (b) atrophic alveoli; (c) fat deposit. X 75. then a wide deviation occurs, classifying them as abnormal. To return, the two strains undergo a similar phenomenon. When the active breeding period ceases (seven to eight months), there is a gradual involution of the mammary tissue. The fat pads (Ing. and Th.) shrink and there is a deposition of pigment, which is often so marked that the adipose tissue takes on a faint yellow tinge. This pigmentation is more noticeable as fibrosis takes place. FEMALE MAMMARY GLAND IN TWO ALBINO MICE 593

Microscopically, the main lactiferous ducts and their branches show a marked increase in the connective-tissue stroma, with hyaline transformation. The lining epithelial cells of the whole system have become atrophic, and a plentiful supply of pigment is scattered among the cells. The lumina of the ducts are often narrowed to such an extent that they are almost obliterated. The encroaching fibrous tissue has pushed the lining cells, on either side, so closely together that they meet in the center.

FIG. 16. SPTM. AGE TWENTYMONTHS. Showing (a) dilated ducts; (b) cyst formation, and increase in connective tissue surrounding ducts and acini; (c) in- guinal deposit. X 75.

In the control strain there is a lack of material in the ducts, but this finding is prominent in the SPTM. The ducts of these animals show an abundant amount of substance, which is homo- geneous in character. There is also cystic degeneration with marked round-cell infiltration throughout these sections. Brownish pigment is diffusely distributed between the epithelial cells and the cells of the fat and connective tissue. The control strain demonstrates nicely the manner in which the gland fibroses with advancing age. The fibrous tissue, which 594 LYDIA M. GIBSON

is hyaline in nature, can be seen creeping out into the fat and replacing the adipose tissue of the deposit. As it contracts, apparently it squeezes the acini until they lose their lumina, so they become nothing but small knots of atrophic cells inter-

FIG.17. CONTROLL. 1. FIFTEENMONTHS. SECTIONTHROUGH IN(~IJINAL DE- POSIT. (a)Fibrosis of deposit, fibrous tissue replacing fat; (b) few ncini ctiuglit in connective tissue; (c) duct (secondary). spersed in the connective tissue. Instead of this phenomenon progressing normally in the SPTM strain, zones of increased epithelial proliferation, both in the duct lining cells and in those FEMALE MAMMARY GLAND IN TWO ALBINO MICE 595 of the alveoli, are evident. This overgrowth is noticeable in specimens showing the picture often referred to as "chronic cystic mastitis." For these are the sections in which the stasis in the ducts is most marked. In some instances, the process has gone on to true neoplastic growth. The acini have lost their

FIG. 18. SPTM. FOURTEENMONTI~R OLD. SECTIONTHROUGH CENTER01" THOHACICDEPOSIT. (a) cyst; (b) cyst with round cell infiltration, increuse in connec- tive tissue surrounding acini. Stagnation of secretion in breast. regular formation and have become carcinomatous. The round- cell infiltration immediately surrounding these areas of meta- plasia is noteworthy (see Fig. 19). These malignant neoplasms are strangely lacking in mitotic figures. There are two types of malignant neoplasms appearing in these animals. One can be classified as a simple adenocar- cinoma, but the other is a more complicated lesion, and bears many of the characteristics of an endothelioma. In the last mentioned neoplasm, the rosette arrangement of the cells, which are small and endothelioid in type, together with the large blood-filled spaces lined with these cells, strongly suggests a tumor of this nature. Most of these neoplasms are confined to 596 LYDIA M. GIBSON

the mammary tissues. They have a tendency to spread out into the fat deposit and to burrow under the corium, but they do not infiltrate the skin to any extent. Surface ulceration is a common occurrence. The thoracic and inguinal portions alike give rise to these growths. Many times the metaplasia has affected several parts of the gland simultaneously. A rare find- ing is a metastatic growt,h in the lymph nodes, most of the

Fro. 19. SPTM. AGE TWENTYMONTHS. Section through inguinnl deposit showing (a) an early malignant neoplasm and (b) a diffuse cystic degeneration of the mammary gland. X 75. Note secretion stagnating in ducts and alveoli.

secondary growths being confined to the lungs or the liver. DISCUSSION:Although the data are limited, certain tendencies stand out when the two strains are compared. The SPTM shows a more rapid and a more complete development of the nipple zones in the embryonic state. But after birth the SPTM shows definite anomalies in the development of the nipples. The control strain shows a more rapid and a more profuse develop- ment of the alveolar tissue up to and through the three month period. No remarkable differences are again noted until the involution FEMALE MAMMARY GLAND IN TWO ALBINO MICE 597

changes accompanying old age are compared. The deposits in the control strain slowly become fibrosed, as the activity of the gland subsides. But in the SPTM, cystic formation is noted. There is also homogeneous material stagnating in the collecting system of the gland, with marked round-cell infiltra- tion in the zones surrounding such areas. Finally, there is early metaplasia of the epithelial cells lining the ducts and the acini

FIG. 20. SPTM. AGE TWENTYTHREE MONTHS.NON-BREEDER. SMALL ADENOCARCINOMA.(a) Level of 6rst inguinal nipple; (b) cystic formation with stasis; (c) alveoli with retained secretion. X 75. of these cystic portions with a later change into the true malig- nant neoplasms, which are characteristically found in the mam- mary glands of this high tumor strain. Bagg's work on the effect of stagnation of secretion in the duct system of the breast and its relation to neoplastic development is interesting to contem- plate in this connection. Also one might theorize on the rBle that anomalies of the nipple play in relation to neoplasia, in this strain of animals. Roger Williams, long ago, noted the ten- dency of athelia and supernumerary to be repeated in certain families, the mother and the daughter show- 598 LYDIA M. GIBSON ing like anomalies. Adair and Bagg think that inverted and under-developed nipples cause obstruction of ducts and stagna- tion within the breast. Six of their series of one hundred women

Flu. 21. SPTM. ACIESIXTEEN MONTHS. MALIGNANT NEOPLASM AT THE LEVELOF THE FIRSTINGUINAL NIPPLE. This is one of the large blood filled cystic tumors which resemble an endotheliomn in configuration, but arise in the mammary deposit. x 75. (a) Blood filled cavity; (b) adenocarcinoma; (c) whorls of cells (endothelioid).

with mrtmmary carcinoma had inverted nipples of one or both . FEMALE MAMMARY GLAND IN TWO ALBINO MICE 599

Bagg found that "prevention of breeding in female mice decreased the incidence of mammary carcinoma and increased the cancer age." The SPTM have a large number of young as a

FIG.22. SPTM. THIRTEENTO SIXTEEN MONTHS OF ACE. ADENOCARCINOMA INVOLVINGREGION BETWEEN Th. 2 AND 3. (a) Primary duct Th. 2, (b) adeno- carcinoma. X 7.5. rule, and are very proliferative. They are often seen suckling large litters (8-9) when they again have become pregnant. When it is recalled that the average life of a mouse is two years, and that the female is mature at three months and that she can 600 LYDIA M. GIBSON have a litter every twenty-one days throughout the active breed- ing periods, some notion of the activity of the mammary gland is attained. After the eighth month, the glands slowly begin to involute. In the SPTM, M. C. Marsh in his paper on "Prob- able Existence of Calendar Rhythm in the Origin of Spontaneous Mammary Tumors in Mice" says, the "expectancy of tumors increases progressively with age . . . there is an unbroken rise in incidence at an almost uniform rate to the tenth month . . . a decrease appears from the eleventh to the thirteenth month." It is also interesting that neither anomalies of the nipples nor stagnation in the ducts was noted in the control animals which belong to a low mammary tumor strain. CONCLUSIONS:1. Although the material is limited, it seems that embryologically the nipple zones in the SPTM are inclined to develop faster than those of the control strain. 2. The SPTM is inclined to show anomalies in nipple develop- ment. 3. The control strain shows a more rapid and a more abundant branching of the lactiferous ducts, and a better alveolar develop- ment up to and through the three-month stage. 4. The control strain has a tendency to have the mammary gland involute and fibrose in an orderly fashion as age advances. 5. The SPTM shows neoplasms developing in zones of chronic cystic mastitis. The epithelial elements of the gland are in- clined to metaplasia rather than atrophy, as age advances.

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