Epithelial induction of stromal tenascin in the mouse mammary gland: From embryogenesis to carcinogenesis

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BIOLOGY

128,245-255 (1988)

Epithelial Induction of Stromal Tenascin in the Mouse Mammary Gland: From Embryogenesis to Carcinogenesis YUTAKA INAGUMA,* MORIAKI KUSAKABE,?’ ELEANOR J. MACKIE,~ CAROLYN A. PEARSON,$ RUTH CHIQUET-EHRISMANN,$ AND TERUYO SAKAKURA+~ *Aichi Cancer Center Research Institute, Ldmatorg of Experimental Pathology, Nagoya 46.4,Japan; TNagoya University School of Medicine, Institute fw Laboratory Animal Research, Nagoya 466, Japan; $Friedrich Mieecher Institute, Post&ch 25J+3,CH-.@02,Base& Switzeda~ and $Tsukuba Life Science Center, Riken, Luboratmg of CeU Biology, Tsukuba, Ibaraki $05, Japan Accepted March Sl, 1988 The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme. Q 1988Academic Press, Inc.

INTRODUCTION

day); formation of the bulbous epithelial structure called endbuds at the tip of the growing ducts at 3 weeks The mouse mammary gland has been widely studied and is a valuable animal model used for experiments in of age; acinar development during pregnancy; preneothe biomedical field. Many papers and reviews on the plastic hyperplastic alveolar nodules and mammary mouse mammary gland are available describing normal tumors; and metastasizing tumors to other organs, in mammary gland development (Turner and Gomez, 1933; particular lung and lymph nodes. Tenascin is a novel extracellular matrix protein origiRaynaud, 1961; Kratochwil, 1986; Sakakura, 1987), hornally described as myotendinous antigen (Chiquet and mone function (Topper and Freeman, 1980), preneoplasia (DeOme et aL, 1959; Medina, 1973; Cardiff, 1984), Fambrough, 1984a,b). In immunohistochemical studies, hormone-responsive tumors (Van Nie, 1967, Van Nie this protein was found to be selectively associated with the mesenchyme immediately surrounding the epitheand Dux, 1971), and neoplasia (Nandi and McGrath, hum of various organs such as mammary gland, tooth, 1973; Hilgers and Sluyser, 1981). In the natural history hair follicle, and kidney during early organogenesis of mouse mammary gland development, from embryo(Chiquet-Ehrismann et aZ., 1986; Aufderheide et al., genesis to carcinogenesis, many steps can be distin1987). Our observations suggest that tenascin is imporguished: formation of mammary streak at lo-11 days, tant for growth promotion. If mammary tumor cells are mammary bud at 13-14 days; sex differentiation of cultured on tenascin-coated plates, they proliferate mammary gland development at 14 days; mammary without serum much more than on tenascin-uncoated sprout at 16 days; penetration of the mammary fat pad pIates (Chiquet-Ehrismann et uL, 1986). The positive precursor tissue showing ductal branching at 17-18 result in tenascin immunohistochemistry of the stroma days during embryonic development (vaginal plug = 0 of malignant tumors supports this possibility (Mackie ’ Present address: The Hospital for Sick Children, Toronto, Canada. et al, 1987). On the other hand, however, tenascin is 2To whom reprint requests should be addressed. absent in the adult rat mammary gland at pregnancy in 245

0012-1606133$3.00 Copyright Q 1998 by Academic Press, Inc. All rights of reproduction in any farm reserved.

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spite of its active proliferation of the acinar epithelium (Chiquet-Ehrismann et ak, 1986). These results lead us to speculate that tenascin does not simply function as a growth factor but may function in a different manner during various phases of mammary gland development. Since tenascin is an extracellular matrix protein, it probably plays an essential role in epithelial-mesenchymal interactions during development. The aim of this study is to elucidate the function of tenascin in mammary gland development and to explore the molecular mechanism of the tissue interactions. The mouse mammary gland is a good tool for this purpose. In the present study, we stained mammary tissues from the mice throughout development as well as various pathological lesions with anti-chicken tenascin rabbit polyclonal antiserum. Tenascin was detected only in the mesenchyme closely surrounding the mammary buds and sprouts of embryos, ductal endbuds of young mice, and malignant mammary tumors. In addition lesions of embryonic resurgence which were induced experimentally in the adult mammary gland by the transplantation of embryonic submandibular mesenchyme (Sakakura et a& 1981) also contained tenascin in their surrounding stroma. The present study provides experimental evidence for the regulation of stroma1 tenascin by the epithelium.

VOLUME 128,1988

glands were obtained from 2- and 3-week-old juvenile, midpregnant (12-14 days of gestation), involuting (5-6 days after removal of the babies), and fully matured 12-month-old virgin female mice. Preneoplastic hyperplastic alveolar nodules (HAN) and mammary tumors were isolated from C3H/HeN+ breeders. Metastasizing tumors were obtained from the lungs of GRWA male mice 4 weeks after the subcutaneous transplantation of the metastatic mammary tumor line GMT-L (see below). A transplanted mammary tumor was obtained from the dorsal subcutis of a BALB/c nude mouse 4 weeks after the injection of 1 X lo6 mammary tumor cells of the CMT315 (see below) cell line. For the present study, GMT-L tumors of the 45th generations and CMT315 cells of the 40th generation were used. GMT-L

GMT-L is a metastatic mammary tumor line. It was established from a spontaneous lung metastasis during serial subcutaneous transplantation of a mammary adenocarcinoma from a GRWA mouse. The tumor line has since been maintained by in tivo passaging for 4’7generations. Transplantation of lung metastasis was performed as follows. Lungs were removed from a mouse carrying a solid tumor of GMT-L on the back, transferred to a MATERIALS AND METHODS sterile plastic petri dish, and minced into small fragments. Several pieces of the tumor fragments were Animals and Tissues syngrafted subcutaneously by surgical incisions on the C3H/HeN+ mice carrying exogenous murine mam- backs of male mice. The tumors grew rapidly reaching 2 mary tumor virus (MuMTV)~ and C3H/HeN- mice freed X 2 cm after 4 weeks at the site of implantation. Lung of exogenous MuMTV by foster nursing on C57BL6 metastases were observed histologically 3 weeks after mothers were mainly used throughout the study. the transplantation and macroscopically 4 weeks after GRWA and BALB/c nude mice were used when needed. the transplantation. Transplantability of GMT-L in the C3H/HeN+ and C3H/HeN- mice were obtained in 1975 subcutis and the metastasizing capacity of the lungs are from the Small Animal Section, Veterinary Resources both 100%. GMT-L tumors are medullary type adenoBranch, Bethesda, Maryland. GRWA mice were ob- carcinomas at both the primary and metastasized sites. tained in 1976 from the Netherlands Cancer InstituteAntoni van Leeuwenhoekhuis Hospital, Amsterdam. CMTSl5 They have been maintained in the animal section of our institute by sister-brother mating. BALB/c nude mice CMT315 is a mammary tumor cell line established were obtained from the Institute for Laboratory Anifrom a mammary tumor which had spontaneously demal Research, Nagoya University School of Medicine, veloped in a C3H/HeN+ mouse. A small adenocarciNagoya. noma was isolated, minced by scissors, chopped by razor The fetuses of specified ages were removed from blade, and digested by 0.1% collagenase (CLS II pregnant mice (vaginal plug = 0 day). The mammary Worthington) in a 25-cm2 culture flask (Falcon) for 1 hr rudiments were isolated from 12- to l&day female fe- in a shaking waterbath at 37’C. The mammary tumor tuses and 14- to 15-day male fetuses. Normal mammary cells were purified by Percoll (Pharmacia) gradient centrifugation (Imagawa et al, 1982) and cultured in Ham’s 3 Abbreviations used: NMU, N-methylnitrosourea; MuMTV, murine F12 culture medium supplemented with 10% fetal calf mammary tumor virus; HAN, hyperplastic alveolar nodule; FCS, fetal serum (FCS Gibco). For passaging, the cells were incucalf serum; PBS, phosphate-buffered saline solution; FITC, fluoresbated in 0.25% trypsin (Difco, 1:250) in phosphate-bufcein isothiocyanate; CSA, CIH-specific antigen; GPI, glucose-phosfered saline solution (PBS), washed twice, and transphate isomerase; DAN, duct-alveolus nodule.

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ferred. CMT315 cultures were passaged every 2 weeks from 1 to 10 plates. For the cloning, the cells were plated in 96-well cluster plates (Falcon) to result in one to eight cells per well and they were maintained in Ham’s F12 with 10% FCS. Once a single colony was visible in a well, it was trypsinized and transferred to a 24-well culture plate (Falcon) and then into a culture flask. The clone which showed the highest tumorigenic capacity when the cultured cells were injected into the young C3H mice was selected. This selection procedure was repeated twice at the 30th and 35th passages. CMT315 cells injected into the subcutis of in C3HiHeN or BALB/c nude mice gave rise to tumors at the site of the injection within 4 weeks with a 100% incidence. These tumors were all medullary adenocarcinomas. Antisera The anti-tenascin antiserum has been described previously (Chiquet-Ehrismann et al, 1986). Preparation of antiserum to CSA will be described elsewhere (Kusakabe et a& submitted for publication). Briefly, the fraction containing glucose-phosphate isomerase (GPI) was collected from muscles and livers of C3H/HeN mice and used as the antigen. For immunization, 400 rg protein emulsified in Freund’s incomplete adjuvant (5:l) was injected into inguinal lymph nodes of (BALB/c X SJL)Fl female mice. Five injections were performed in the same way at monthly intervals. Antibody production was assessed by using immunochemical staining of both primary cultured cells and histological sections obtained from C3H mice. Immunohistochemistrg Five to 10 mammary tissues were investigated for each experiment. The results of these samples were all the same within the groups analyzed, Tissues were fixed in 10% buffered formalin. After washing and dehydration, they were embedded in polyester wax, sectioned, and stained by indirect immunofluorescein method according to the procedure described by Kusakabe et al. (1984). The first antibody was anti-chicken tenascin rabbit antiserum, diluted ZOO-fold in PBS. The second antibody was fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit immunoglobulin (Bio-Yeda). For the staining with antibody to the C3H-specific antigen (CSA) the FIT&labeled avidin-biotin technique was used; the sections were incubated with biotinylated antibody for 30 min, washed three times with PBS, and incubated with FIT&conjugated avidin for 30 min. After the immunohistochemical studies by fluorescence microscopy all sections were stained with hematoxylin and eosin for histological observation.

Wholemount Preparation C3I-I female mice were killed at 2 and 3 weeks after birth and fixed in 10% formalin. The inguinal mammary glands were removed from the skin, pressed between two glass plates, defatted by acetone, washed, stained by hematoxylin, dehydrated, and embedded in Canada balsam. The morphology was examined under a dissecting microscope. Transplantation of Embryonic Salivav Gland Mesenchyme into Adult Mammary Gland GRS/A mice were used for these experiments. The salivary gland rudiments were dissected from 13- and 14-day embryos of both sexes and were transplanted into the No. 4 inguinal mammary glands of both sides of 2-month-old virgins by the technique described previously (Sakakura et al, 1979). In response to the embryonic salivary gland mesenchyme the adult mammary epithelium starts to proliferate in multiple layers and 2-4 days after implantation resembles embryonic rudimental architecture. Then, ductal branching occurs, followed by the formation of nodular hyperplasia of ducts and alveoli termed duct-alveolus nodules (DAN) (Sakakura et al, 1981). At autopsy DANs were recognizable macroscopically as pinkish lesions at 7 days after transplantation and as white, translucent, solid nodules at later stages. DANs obtained from GRS/A mammary glands ‘7 and 14 days after the mesenchyme transplantation were fixed in 10% buffered formalin for immunohistochemical studies. Culture of Embryonic Mammary Epithelium C3H/HeN- mice were used for these experiments. Details of the procedure have been described elsewhere (Inaguma et aZ., 1987). Fat pads were removed from 14-day embryos, minced, explanted in eight chamber slides (Lab Tek) containing Ham’s F12 culture medium with 10% fetal calf serum, and incubated at 3’7°C in a 5% COZ absorption. The mammary epithelia were isolated from the mammary glands of 17-day female embryos by collagenase digestion. The fat pads were cultured for 2-3 days, then one to three pieces of the mammary epithelia were placed on them, and 3-5 days later the cultures were fixed in 10% formalin. After washing with water, they were stained by anti-tenascin antiserum for 3 hr and then with FIT&labeled goat antirabbit immunoglobulin for 3 hr by the methods described above. RESULTS

Embryonic Mammary Gland In 12-day embryos, the mammary gland rudiment forms a lens-shaped mammary bud composed of several

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layers of epidermal cells. The mesenchymal cells are present diffusely under the epidermal layer without forming any recognizable structure. This is the first outwardly visible evidence of mammary gland development. At this stage no tenascin was detectable (Fig. 1). At the 13th day of gestation, the mammary rudiments start to change from lens-shaped to bulb-shaped with a narrow neck. Immunofluorescent staining for tenascin was weakly positive in the extracellular matrix of the mesenchyme closely surrounding the mammary epithelium. The sexual phenotype of the mammary gland is determined on Day 14. In the female mammary gland, a dense mammary mesenchyme is clearly observed as several layers of fibroblasts surrounding the mammary epithelium (Fig. 2). In the male mammary gland, this mesenchyme constricts the mammary bud causing the subsequent rupture of the mammary rudiment (Fig. 3). At this stage tenascin staining was stronger than at Day 13 in both male and female mammary glands. At the late 16th day, the mammary bud proliferates, rapidly elongating and forming mammary sprouts (Fig. 4). It grows downward penetrating the mammary fat pad precursor tissue and branches at the 17th day (Fig. 5). Tenascin was seen in the dense mesenchymal cells surrounding the mammary epithelial stalk but not in the mesenchymal cells of the distinct fat pad precursor tissue surrounding the branching ducts.

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Hllperplastic Alveolar Nodule (HAN)

HAN is a preneoplastic mammary lesion which is similar in morphology to the mammary gland during alveolar development in mice in mid-to-late pregnancy. The alveolar cells of HAN proliferate with secretory activity, and they are surrounded by very little fibroblastic stroma. These fibroblasts were not stained by anti-tenascin (Fig. 12). Mammary Tumor

In C3H mice, almost all mammary tumors are histologically classified as Type A or B (Dunn, 1953).Type A mammary tumors are well-differentiated adenocarcinomas showing acinar structure. Type B mammary tumors are undifferentiated adenocarcinomas showing papillary or intracanalicular growth and cyst formation. Tenascin was present in the stroma of all these mammary tumors, particularly in the areas apposing the tumor epithelium (Figs. 13 and 14). Tenascin was also detected in the lung metastases of mammary tumors (Fig. 15). The tissue distribution of tenascin during morphogenesis and carcinogenesis of the mammary gland is summarized in Scheme 1 and Table 1. Duct-Alveolus Nodule

DAN is a hyperplastic lesion experimentally induced in the adult mammary gland by transplantation of embryonic submandibular gland mesenchyme (Sakakura Juvenile Mammary Gland et ak, 1981). Seven days after transplantation, the host As demonstrated in the whole-mount preparations epithelial cells in contact with the submandibular mesthe morphology of the mammary gland of 2-week-old enchyme proliferate in multiple layers and develop juvenile mice is different from that of the mammary ductal buds resembling the embryonic mammary rudigland of 3-week-old mice (Figs. 6 and 7). The branching ments. Tenascin was detected in the mesenchyme surpattern and the area of the fat pad they occupy are rounding these ductal buds, but not in the other part of similar but the mammary ducts are thicker in the 3- the transplanted submandibular gland mesenchyme week-old mammary gland than in the e-week-old mam- (Fig. 16). Two or more weeks later, the mammary epimary gland. Many endbuds have formed at the tip of the thelium responding to embryonic submandibular mesducts of 3-week-old mammary glands. Tenascin stain- enchyme undergoes alveolar development and produces ing was negative in the 2-week-old mammary gland a localized nodule. At this stage tenascin was no longer (Fig. 8), while in the 3-week-old mammary gland tena- observed (Fig. 17). scin appeared in the mesenchyme surrounding the newly formed endbuds (Fig. 9). Then as the endbud Induction of Stromal Tenascin Expression by Explantation of Epithelial Cells elongated, tenascin disappeared from the tip and was present only in the neck of the endbud (Figs. 10 and 11). Subcutaneous injection of the CMT315 cell line originating from C3H mice into BALB/c nude mice gave rise Fully Matured, Pregnant, and Involuting to tumors. In an immunohistochemical study of these Mammam Glands tumors, tenascin was detected only in the stroma immeMammary glands of 12-month-old virgin females diately surrounding the mammary tumor and between contained no tenascin. During pregnancy acinar prolif- alveolar nests. Tenascin was not present in the general eration takes place in the mammary glands and after subcutis (Fig. 18). In sections stained with antiserum to removal of the weanlings acinar involution occurs. the C3H-strain-specific antigen CSA, all epithelial cells were stained positively but the fibroblasts within and Tenascin was not detected at any of these stages.

FIIGS. l-5. Distribution of tenascin in embryonic mouse mammary glands. Tenascin appears on the 14th day in the dense mammary mesemchyme immediately surrounding the epithelia in both male and female embryos, but is absent in the elongating ducts of 17-day embryos. (4 1.ndirect immunofluorescence polyester wax sections of 13-day female (l), ll-day female (2), 15-day male (3), Is-day female (4), and 17-day femaJe (5) embryonic mammary glands. (B) The same sections shown in (A) stained by hematoxylin and eosin. Bar, 100 pm. 249

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Develoomentol II-d

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Tenascin in Mammary Gland

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of Tenoscln

In 14-d

embryo

\

Mclm83rY

Gland 18-d

embryo

Tissue

embryo

I Fot Pod Precursor Tissue

Juvenile n-

MomnotyTunor

SCHEME1. 0, Tenascin staining; E, mammary epithelium; 1, tenascin surrounding terminal ducts (Fig. 9); 2, tenascin concentrated on the flank of the endbud (Fig. 10); 3, Tenascin concentrated in cleft of bifurcating endbud (Fig. 11).

surrounding the tumor were not stained, indicating clearly the C3H origin of the tumor cells and the BALB/c origin of the tumor stroma (Fig. 19). The donor epithelium therefore induced the host stroma to lay down a tenascin-containing matrix. The induction of tenascin by epithelium was further verified in vitro using the culture system of embryonic mammary glands on the fat pad feeder layers described recently (Inaguma et aL, 1987). The explanation of embryonic epithelial mammary rudiments onto mesenchyma1 fat pad feeder layers led to the accumulation of tenascin in the extracellular matrix of the feeder cells closely surrounding the embryonic epithelium (Fig. 20). DISCUSSION

We investigated the distribution of the extracellular matrix protein tenascin during normal and abnormal mammary gland development. We found that tenascin first appeared at the late 13th day of gestation in the mesenchyme immediately surrounding the early mammary epithelium. Tenascin was no longer present in the region of epithelial development at the 17th day. How-

ever, once the reproductive system started to function at about 3 weeks of age, tenascin was present in the mesenchyme surrounding endbuds. Endbuds are bulbous structures at the tips of the growing ducts. They develop as a result of intense mitotic activity (Russo and Russo, 1978; Williams and Daniel, 1983) and they are thought to contain the mammary stem cells (Dulbecco et uL, 1986; Sonnenberg et al, 1986) which proliferate and differentiate in response to hormones. Tenascin was not detected around proliferating acini at pregnancy nor around preneoplastic HANS, lesions of the mammary gland resembling morphologically the pregnant mammary gland. However, once the mammary epithelium was transformed into malignant epithelium the surrounding mesenchyme started to express tenascin. Thus, in the course of mammary gland development from embryogenesis to carcinogenesis tenascin appears only in the mesenchyme immediately surrounding the epithelium just starting morphogenesis and growth. However, the absence of tenascin in the proliferating acini of pregnant mice and in preneoplastic HANS clearly indicates that tenascin is not required for mere

FIGS. 6 and ‘7.Wholemounts of 2-week old (6) and 3-week-old (7) female mouse mammary glands. Many endbuds (eb) are formed at the tip of the ducts of 3-week-old mammary gland stained by hematoxylin. Bar, 500 pm. FIGS. 8-11. Distribution of tenascin in juvenile mouse mammary glands. Tenascin is absent in the mesenchyme of e-week-old mammary gland (8) but present in the mesenchyme surrounding the whole endbud (9,10) and then the neck of the branching endbud (11) in 3-week-old mammary gland. (A) Indirect immunofluorescence of polyester wax sections. (B) The same sections shown in (A) stained by hematoxylin and eosin. Bar, 100 pm.

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TABLE 1 TENASCINLOCALIZATIONWITH REGARDTO GLAND GROWTH Glandular

growthstate

Embryonic Day 12 Day 13 Day 14 Days 16-17 Juvenile Week 2 Week 3 Week 3 Adult Month 3 Pregnant Involuting HAN Adenocarcinoma A, B Lung metastasis DAN * -, no stained, t, weakly stained, tt,

Epithelial growth

Tenascin localization

-

t

-* +* ++* Surrounds epithelium t + Stalk only

+

+t Surrounds endbud -t-t Endbud flank only

-

(Fig. 1) (Fig. 2) (Figs. 4 and 5) (Fig. 8) (Fig. 9) (Figs. 10 and 11)

-

t t + + +

++

Surrounds epithelium tt Surrounds epithelium + Surrounds epithelium

(Fig. 12) (Figs. 13 and 14) (Fig. 15) (Fig. 16)

strongly stained.

proliferation of epithelial cells. The same phenomenon was also demonstrated in the experimentally induced morphogenesis in adult mammary glands. If embryonic submandibular gland mesenchyme was transplanted into the mammary gland of adult mice, the mammary epithelium responded by developing endbud-like structures, and then forming a duct-alveolus nodule which histologically resembled a HAN (Sakakura et ak, 1979, 1981). Tenascin was present initially in the connective tissue surrounding the new endbuds but was not present in the duct-alveolus nodule at the later stage. In a previous study (Chiquet-Ehrismann et aL, 1986), we found that tenascin supports the growth of cultured mammary tumor cells, after the removal of fetal calf serum, much better than other extracellular matrix proteins such as fibronectin, laminin, and type I and IV collagens. In the present study, tenascin was shown to

be present in the mesenchyme surrounding the epithehum starting to develop morphogenetically, but not in the cellular proliferation leading to cytodifferentiation during pregnancy and preneoplastic development. Taking these results together, we conclude that tenascin is not generally needed for epithelial proliferation but is essential for the early stages of the morphogenetic process. In fact, when embryonic mammary epithelium is recombined with the embryonic dense mammary mesenchyme in which tenascin is detected by immunohistochemistry and transplanted under the kidney capsule, the mammary epithelium forms a ductal hyperplasia. If, on the other hand, the mammary epithelium is recombined with the other part of the mammary gland mesenchyme (fat pad precursor tissue) which is not stained by anti-tenascin, the resulting gland shows the normal mammary morphogenesis (Sakakura et aZ.,

FIGS. 12-15. Distribution of tenascin in mammary neoplasia. Tenaacin is not present in the mesenchyme of preneoplastic HAN (12, nonspecific staining is seen in the secretion of alveoli) but is present in the dense mesenchyme of type A (13) and B (14) mammary tumors which developed spontaneously in C3H mice. Tenascin also appears in lung metastases (15). (A) Indirect immunofluorescence of polyester wax sections. (B) The same sections shown in (A) stained by hematoxylin and eosin. Bar, 100 pm. FIGS. 16 and 17. Distribution of tenascin in l-week (16) and 3-week (17) DANs induced by the transplantation of embryonic. submandibular gland mesenchyme. Tenascin is detected in the mesenchyme surrounding ductal buds of l-week DAN but not in the proliferating ducts of a-week DANs. (A) Indirect immunoflurescence of polyester wax sections. (B) The same sections shown in (A) stained by hematoxyiin and eosin. Bar, 100 pm. FIGS. 18 and 19. Distribution of tenascin and CSA in the tumor induced by subcutaneous injection of CMT315 cells into a Balb/c nude mouse. Tenascin is detected only in the mesenchyme immediately surrounding the tumor and between alveolar nests but is not detected in the general subcutis (SC)(18). These mesenchymal cells are not stained by antiCSA, indicating clearly the Balb/c origin, while epithelial cells are stained by anti-CSA indicating the C3H origin (19). (A) Indirect immunofluorescence of polyester wax sections stained by anti-tenascin (18) and anti-CSA (19). (B) The same section shown in (A) stained by hematoxylin and eosin. Bar, 100 pm. FIG. 20. Distribution of tenascin in mammary morphogenesis in vitro. Tenascin is detected only in dense mesenchyme surrounding the cultured mammary epithelium. The other mesenchymal cells of the feeder layer are not stained by anti-tenascin. (A) Indirect immunofluorescence of the whole culture tissue. (B) The same tissue by phase-contrast microscopy. Bar, 1 mm.

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1982).Thus, in mammary gland development tenascin is likely to be an essential factor in the epithelial-mesenchymal interactions preceding the morphogenetic development of the gland-specific branching pattern. Silberstein and Daniel (1982) have found that in the growing endbud the flank region and the cleft between branches are rich in sulfated glycosaminoglycans. They speculated that these molecules play a role in establishment of interductal spacing possibly by collagen fibrillogenesis in the stroma. In the present study we demonstrated the tenascin expression at the same sites of the endbuds. Since these sites are less actively growing and relatively differentiated, there may be a clear association of tenascin with the process of morphogenetic stabilization by sulfated glycosaminoglycans. At present, the interactions between tenascin and other extracellular matrix substances are not elucidated yet. Chiquet and her associates (1988) have only demonstrated that tenascin inhibits mammary tumor cell attachment to fibronectin substrate and promotes the release of single cells from epithelial cell sheets in vitro. The function of tenascin would thus be an inhibition of the cell attachment and growth promotion for the cultured cells. While histological studies of the mammary glands of embryos and juveniles indicate somewhat growth-inhibiting function. The explanation of this discrepancy is of fundamental interest in our laboratory. An important question arises as to whether tenascin appears as a cause or a consequenceof the interaction between epithelium and mesenchyme. Several observations indicate that tenascin appears in the mesenchyme as a result of the inductive interaction with the epithelium of certain developmental lineages. First, tenascin was detected at the site where the first mammary tumor cell(s) metastasized and formed a small nest in the lung. Second, when the C3H mammary tumor cell line CMT315 was transplanted into BALB/c nude mouse we could show that only the epithelium of the developing tumors was stained by anti-cross-strain marker CSA and that the surrounding connective tissue producing tenascin was of BALB/c origin. This is clear evidence that the malignant epithelium induced the tenascin in the surrounding host stroma. Third, the induction of tenascin in stromal cells by embryonic epithelium was verified in vitro. When mammary fat pad precursor tissue was cocultured with embryonic mammary epithelium, the mesenchymal cells closely surrounding the epithelium condensed, and tenascin was detected in these condensed cells but not in the mesenthyme at the peripheral site. Epithelial-mesenchymal interactions are very important in organogenesis and organostasis in embryos as well as in adults (Grobstein, 1967; Kratochwil, 1972; Cunha et aL, 1985). During the development of many

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organs, reciprocal interactions between epithelium and mesenchyme occur sequentially. As a result of those multistep interactions, the epithelial and mesenchymal cells acquire higher levels of differentiation. In mammary gland development, the chain of tissue interactions has not been elucidated yet. The dense mammary mesenchyme determines the mammary epithelium’s ability to interact with the fatty stroma (Propper, 1968; Sakakura, 1983). The mammary epithelium in turn induces androgen receptors in the mammary mesenthyme (Heuberger et al, 1982), and then the mesenthyme condenses in response to endogenous androgen and causes degeneration of the male mammary gland (Kratochwil, 1971; Kratochwil and Schwartz, 1976; Drews and Drews, 1977; Diirnberger and Kratochwil, 1980;Heuberger et al, 1982;Wasner et al, 1983). In the present study, we have demonstrated another link in the chain of reciprocal interactions between epithelium and mesenchyme. Mammary epithelium of certain developmental stages during embryogenesis and neoplasia induces tenascin synthesis in the mesenchyme, and in turn the mesenchyme probably induces the growth and morphogenesis of the epithelium. Further study on the mechanism of tenascin appearance is our next project. We thank Dr. Howard L. Hosick for many helpful discussions. This work was supported by the Ministry of Education, Science, and Culture of Japan. REFERENCES AUFDERHEIDE, E., CHIQUET-EHRISMANN,

R., and EKBLOM, P. (1987).

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