Localization of thrombospondin, CD36 and CD51 during prenatal development of the human mammary gland

Differentiation (1994) 57:133-141

Differentiation

Ontogeny, Neoplasia dnd Differentiation Therapy

0 Sorineer-Verlae 1994

Localization of thrombospondin, CD36 and CD51 during prenatal development of the human mammary gland C. PCchoux'.', P. Clezardin3,R. Dante4, C.M. Serre3,M. Clerget', N. Bertin2, J. Lawler5, P.D. Delmas 3 , J.L. Vauzelle ', L. Frappart

' Laboratoire d' Anatomie Pathologique, Bat 10 Hopital Edouard Herriot, F-69437 Lyon, France ' UnitC CNRS UPR-4 12, Passage du Vercors, F-69367 Lyon, France

' INSERM Research Unit 403, Pavillon F, Hopital Edouard Herriot, F-69437 Lyon, France INSERM Research Unit 218, 28 rue Laennec, F-69373 Lyon, France Vascular Research Division, Brighani and Women's Hospital, Boston, MA 021 15, USA Accepted in revised form February 25, 1994

Abstract. Thrombosporidin (TSP) is a 450 kDa extracellular matrix glycoprotein expressed in normal, hyperplastic, and neoplastic human breast. In this study, the patterns of expression of TSP were determined during development of the human fetal mammary gland between the 15th and the 39th week of gestation. Using immunohistochemistry, TSP is found in the dense mesenchyme immediately adjacent to the mammary bud, and at the membrane of budding epithelial cells invading the surrounding mesenchyme. As formation of the ductal tree system occurs, TSP is deposited at the myoepithelial-stromal junction of mammary ducts. Such an immunolocalization of TSP in buds and ducts of the fetal mammary gland has been confirmed at the mRNA level using in situ hybridization. Presence of TSP transcripts in nascent breast tissue has been also demonstrated by polymerase chain reaction assay. Comparison of TSP immunolocalization with that of two known TSP cell surface receptors, CD36 and CD5 1, reveals no codistribution of TSP with these receptors during mammary gland development. As opposed to TSP, CD36 is strongly expressed at the membrane of preadipocytes present in the fat pad tissue, but absent from budding epithelial cells. CD51 is only weakly expressed by malpighian epithelial cells and does not colocalize with TSP. In lactating ducts of a newborn, TSP disappears from the myoepithelial-stromal junction of ducts and is synthesized at the apices of secretory epithelial cells of lactating ducts together with CD36. In conclusion, our findings support the existence of an important role for TSP during development of the human fetal mammary gland. Ultimately, variations in the appearance and distribution of TSP and CD36 during the different steps of the mammary gland developmental process may lead to changes in TSP functions, resulting in migration of budding epithelial cells or differentiation of ductal epithelial cells.

Correspondence to: L. Frappart, UnitC CNRS UPR-412, Cytologie MolCculaire, Passage du Vercors, F-69367 Lyon, France

Introduction During the 4th week of embryonic life, development of the human mammary gland is first observed by enlargement of a single epithelial layer of the ectoderm: the mammary streak [32]. This becomes the mammary bud when epithelial cells start to invade the underlying dense mesenchyme [32]. Mammary buds then elongate into mammary sprouts while epithelial cells invade the fat pad precursor tissue [32]. This is followed by an initial branching of mammary sprouts which occurs between the 13th and 20th week of gestation [32]. The basal surface of the growing end sprout is constituted by a layer of cap cells which could then contribute to the formation of the ductal tree system of the mammary gland [ 151. There is increasing evidence that migration, proliferation and differentiation of many cell types can be affected by the extracellular matrix to which they are exposed [5]. Despite the growing recognition of the importance o f this extracellular matrix, few data are available on the pattern of synthesis and deposition of extracellular matrix components and other cell surface constituents during mammary gland morphogenesis, especially in humans. To date, laminin [34], glycosaminoglycans [35] and type IV collagen [34] have been shown to be present in the basement membrane lying between the epithelium and the mesenchyme of the embryonic mouse mammary gland. As mouse mammary epithelial cells start to invade the fat pad precursor tissue, fat pad precursor cells initiate synthesis of laminin, type IV collagen and heparan sulfate proteoglycans [19, 341. Fibronectin [34], type I and t y p e V collagens [42], and entactin [43] are associated with the mesenchyme of the embryonic murine mammary gland. Tenascin is selectively observed in the condensing mesenchyme near budding epithelial cells [9], while it disappears during sprouting o f mouse mammary epithelial cells [18]. Thrombospondin (TSP) is a 450 kDa extracellular matrix glycoprotein synthesized and secreted by a wide range of normal and transformed cells [lo]. Recently, five distinct genes encoding for four structurally different

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TSPs (TSP1, TSP2, TSP3 and TSP4), and cartilage oligomeric matrix protein (COMP) have been described [ 31. The functions of TSP2, TSP3, TSP4, and COMP are unknown. During organogenesis of mouse embryo, there is precise regional and temporal appearance of TSP in tissues (central nervous system, heart, liver, muscle and bone), followed by a disappearance as differentiation proceeds [ 14, 29, 301. In vitro, TSPl supports adhesion 124, 39, 41, 441, migration [26, 37, 381 and proliferation [ I , 2.5, 3 11 of various cultured cells. These functions are mediated through various cell surface receptors including heparan sulfate proteoglycans [14, 361, CD36 [4, 271, CD5 1/CD61 (the a,P1 integrin) [24], a 105/80 kDa cell surface receptor [44], and a 50 kDa protein [40]. Not all of these interactions occur i n all cells, and it is possible that separate cell surface receptors are used in a cooperative manner to bind TSP [4]. We have previously reported that TSP, CD36 and the a, subunit integrin (CDS1) are highly expressed in subpopulations of invasive human breast carcinoma cells [ 131. These findings, taken together with the fact that strong similarities are observed between embryonic development and malignant invasion, prompted us to examine the patterns of expression of TSP, CD36 and CD5 1 during development of the human fetal mammary gland between the 15th and the 39th week of gestation.

Methods Tissue Human mammary glands were obtained from seven male and eight female normal aborted fetuses aged between 15 and 39 weeks, snap-frozen in liquid nitrogen and stored i n liquid nitrogen until used. Anribodies. Mouse monoclonal antibodies P I0 and MA-I are directed against human blood platelet TSPI. Antibody P10 is directed against the type 2 repeats, and the epitope for MA-I is in the last type 3 repeats of platelet T S P l . The characterization and specificity of these antibodies have been described earlier [ I 1 , 231. Because of the high homologies between TSPl and TSP2 in the type 1 and type 2 repeats, the antigen imniunodetected by P I 0 and MA-I was named TSP. Mouse monoclonal antibodies FA6-IS2 and OKM5, which are directed against CD36, were purchased from Immunotech (Lurniny, France) and Ortho Diagnostics System (Paris, France), respectively. Mouse monoclonal antibody LM 142 [8], which is directed against the a, subunit integrin (CD51), was kindly provided by Dr. D. Cheresh, Scripps Clinic and Research Foundation, La Jolla, Calif., USA. Mouse monoclonal antibody P1E6, which is directed against the a2subunit integrin (CDw49b), was purchased from Telios Pharmaceuticals (San Diego, Calif., USA). Mouse monoclonal antibody MARK I , which is directed against rat k-light chains, was purchased from Immunotech (Luminy, France). Rabbit anti-mouse IgG was obtained from Dakopatt (Copenhagen, Denmark). Goat anti-rabbit IgC conjugated with horseradish peroxidase was obtained from Clonatech (Paris, France). /mmicnohi,srochemistryy.The procedure was essentially as described previously [ 131. Frozen sections (5-8 pm thick) were cut in a cryostat at -20" C, air dried, and fixed i n cold acetone. After rehydration in phosphate-buffered saline (PBS), pH 7.2, endogenous peroxidase activity was blocked by a 15 min incubation with S % (vol./vol.) hydrogen peroxide in PBS. Tissue sections were then incubated with the primary antibody previously diluted at a concentration of 0. I pg/ml in PBS containing 1 % (masslvol.) bovine serum albumin. At this stage, a negative control was included

Fig. 1. Topographical changes of epithelial cells during mammary gland development of a 19-week-old female fetus. As observed by light microscopy, the malpighian epithelium ( M E ) enlarges at the future nipple site to form a mammary bud ( M B ) . Budding epithelial cells invade the surrounding dense mesenchymc ( D M ) and sprout to constitute the ductal tree system ( D ) within the fat pad tissue ( F P ) . Bar, 30 pm

Fig. 2. A Mammary bud of a 24-week-old female fetus immunostained for thrombospondin (TSP); the immunoreactivity is found i n the dense mesenchyme immediately adjacent to the mammary bud ( M B ) .Note increased TSP deposits (arrows) at the tip o f a mammary bud which starts to sprout. B Mammary sprout of a 24-week-old female fetus immunostained for TSP. As for mammary bud, TSP is found i n the dense mesenchyme underlying the basal surface of epithelial cells, and a stronger immunostaining is always found where epithelial cells are invading the mesenchyme (arrow). C Mammary duct of a 19-week-old female fetus immunostained for TSP. TSP is seen as a thin pale line i n the basement membrane surrounding the mammary duct (arrows). The vascular endothelium of dermic vessels (arrowheads) is also strongly immunoreactive for TSP. D Mammary bud of an 18-week-old female fetus immunostained for CD36; superficial cells of the malpighian epithelium (*) and the endothelium (arrowhead) are immunoreactive for CD36, while budding epithelial cells are negative ( a r r o w ) . E Mammary sprout of an 18-week-old female fetus immunostained for CD36; the immunostaining pattern is similar to D. F Mammary ducts of a 18-week-old female fetus immunostained lor CD36; immunoreactivity is only observed in endothelial cells ( u r r o w . ~and ) fibroblasts (arrowhead). Bars, 10 pm

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I36 using monoclonal antibody MARK I (0.I pg/ml). After 30 min incubation, tissue sections were washed and incubated for 30 min with a rabbit anti-mouse IgG [diluted to 1 : 80 in PBS containing 25 % (vol./vol.) human serum AB]. After washing, slides were incubated for 30 min with a goat anti-rabbit IgG conjugated with horseradish peroxidase [diluted to 1: 80 in PBS containing 25 % (vol./vol.) human serum AB]. The washing procedure was repeated, and the reaction was revealed with a solution of 10 mg of 3,3' diaminobenzidine tetrahydrochloride in 10 ml 0.05 M TRIS buffer, pH 7.6, containing 10 pl of 30 % H,O,. The reaction was stopped by washing in tap water. Sections were then briefly counterstained with Mayer's hematoxylin, dehydrated in graded alcohols, cleared in methyl cyclohexane and mounted. Sections were examined and photographed using a Leitz Dialux 20 EB photomicroscope.

PoI.ytnerase chain reaction (PCR) assay. Total RNA was prepared from a frozen pulverized sample by the RNAzol method (Bioprobe Systems, Paris, France). Detection of TSP transcripts was carried out as described by Fuqua et al. [ 161, with minor modifications. Briefly, reactions were performed with 0.1 pg of RNA in loop1 of a buffer 110 mM TRIS-HCI (pH 8.3), 1.5 mM MgCI,, 50 mM KCI] containing 0.1 mg/ml gelatin, 80 p M of each of the deoxyribonucleoside triphosphates, and 0.25 p M of each of the TSPl primers (GAG TTG CAA GCC ATG TGC GGC and GGC AGG ACA CCT TTT TGC AGA). After initial denaturation for 2 min at 92" C, I 0 units of M-Mulv reverse transcriptase (Boehringer Mannheim, Meylan, France) were added to the reaction mixture which was then incubated for 35 min at 42" C. After incubation, reverse transcriptase was heat-inactivated (3 min at 94" C ) and, after cooling to 0" C, PCR amplification of the cDNA product was carried out by adding 5 units of Taq I DNA polymerase (Boehringer Mannheim, Meylan, France). PCR amplification was done using 35 cycles in an Eppendorf thermocycler in the following conditions: denaturation for I min at 94" C , annealing for 2 min at 55" C and extension for 3 min at 72" C. Control experiments were performed for each RNA sample, omitting reverse transcriptase, to confirm that the hybridization signal resulted from specific amplification of the RNA, and not of the DNA. Extracted PCR products were then analyzed on a 2 % agarose gel containing 0.1 pg/ml of ethidium bromide.

In situ hybridization Preparcition of ti.tsue sections. Nascent breasts were aseptically removed and fixed overnight at 4" C in PBS containing 4 % (mass/ vol.) paraformaldehyde. Fixed tissues were then dehydrated in graded alcohols prior to embedding in paraffin. Tissue sections (5 pm) were cut, adhered to glass slides previously treated with 3-amino propyltriethoxysilane (Sigma, Isle d' Abeau, France) and then stored at 4" C until used. Preprution of RNA probes and in situ hybridization. Human TSP cDNA clone M9 specifically codes for the NH,-terminus of TSPl [A(-15) to W ( 3 5 5 ) ] [22]. The cDNA was inserted into the EcoR 1 site of pGEM-2 (Promega Biotec, Charbonnikres, France) and used to derive anti-sense and sense probes. RNA probes were generated using l 0 0 p C i a7'S UTP, and either SP6 or T7 polymerases. The pGEM-2/TSP construct was linearized with BumH 1 and transcribed with SP6 RNA polymerase (Boehringer Mannheim, MeyIan, France) to obtain an anti-sense probe. Alternatively, the pGEM2/TSP construct was linearized with Sphl and transcribed with T7 RNA polymerase to obtain a sense probe. Tissue sections were dewaxed and treated with proteinase K (5 pg/ml) for 30 min at 37" C. After proteinase K treatment, sections were washed twice in PBS (2 midwash) and dehydrated by graded concentrations of ethanol. Tissue sections were then processed for in situ hybridization as previously described [ 131.

Results During development of the human mammary gland, epithelial cells enlarge and form a bulb-shaped structure: the mammary bud. The mammary bud elongates by rapid cellular proliferation, forming the mammary sprout which then invades the fat pad precursor tissue. This is followed by an initial branching of the mammary sprout which forms the ductal tree system of the mammary gland. Figure 1 illustrates the topographical changes of epithelial cells during mammary gland development. Using immunohistochemistry and in situ hybridization, the expression of TSP and its receptors (CD36, CDS1) were therefore studied during the different steps of the mammary gland developmental process.

Immunohistochernistry Nascent breast tissue sections of human fetuses (seven males and eight females) aged between 15 and 39 weeks were examined by immunohistochemistry using mouse monoclonal antibodies directed against TSP, CD36, CDS 1 (the a, subunit integrin), CDw49b (the a, subunit integrin) and MARK-1, a negative control mouse monoclonal antibody directed against rat K light chains. Mouse monoclonal antibodies P10 and MA-I gave a similar immunostaining pattern although a stronger immunoreactivity was always observed for MA-I. No immunostaining was observed when tissue sections were examined with MARK1. TSP immunoreactivity was found in the dense mesenchyme immediately adjacent to the mammary bud (Fig. 2 A). Moreover, increased TSP deposits were consistently observed at the tip of the growing end bud. Figure 2 A shows this increased TSP deposit at the tip of a mammary bud which starts to elongate to form a mammary sprout. Similarly, in the mammary sprout, TSP was present in the dense mesenchyme underlying the basal surface of epithelial cells, and a stronger immunostaining was always observed at the tip of the mammary sprout where epithelial cells are invading the mesenchyme (Fig. 2 B). Finally, TSP was weakly but consistently visible as a thin pale line in the basement membrane surrounding fetal mammary ducts (Fig. 2 C). In addition, the vascular endothelium of dermic vessels was strongly immunoreactive for TSP. Budding and sprouting mammary epithelia1 cells were negative for CD36, while superficial cells of the malpighian epithelium were strongly immunoreactive (Fig. 2 D and 2 E). Similarly, no CD36 immunoreactivity in either myoepithelial or luminal epithelial cells was observed in fetal mammary ducts (Fig. 2 F). However, a moderate-to-strong staining for CD36 was consistently observed in endothelial cells and fibroblasts surrounding ducts (Fig. 2 F). As opposed to TSP (Fig. 3 A ) , a strong staining for CD36 was observed at the plasma membrane of preadipocytes which were present in the fat pad precursor tissue (Fig. 3 B). Interestingly, in one case of newborn female (39th week of gestation), the apices of secretory epithelial cells of lactating ducts (witch's milk) were

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Fig. 3. A Fat pad tissue of an IS-week-old female fetus immunostained for TSP; no reactivity is observed. B Fat pad tissue of an 18-week-old female fetus immunostained for CD36; strong staining is observed at the plasma membrane of preadipocytes. C and D

Mammary gland of a newborn female immunostained for TSP and CD36, respectively. Both TSP and CD36 become localized at the apical surface of luminal epithelial cells (arrows), while the basement membrane is completely negative (arrowhead). Bars, 10 pm

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Fig. 4. A Mammary sprout of a 28-week-old male fetus immunostained for CD5 I ; a weak staining is observed on the basal (arrows) and parabasal (arrowheads) malpighian epithelial cells. B Mammary sprout of a 28-week-old male fetus immunostained for

1

2

CDw49b; a moderate-to-strong staining is observed on basal (nrrows) and parabasal (arrowheads) malpighian epithelial cells and on sprouting epithelial cells. Bar.s, 10 km

sue (not shown) were weakly labeled. By contrast, as early as the 17th week of gestation, a strong immunoreactivity for CDw49b was observed in mammary epithelial cells associated with bud, sprout and ducts, as well as in endothelial cells and in preadipocytes (Fig. 4 B). RNA polymerase chain reaction (PCR) assay

Fig. 5. ldentification of TSPl transcripts in nascent human breast tissue using polymerase chain reaction (PCR) assay. Lanes 1 and 4: molecular weight markers. Lane 2: electrophoresis of the 268 bp PCR product. Lanp 3: partial restriction map of the 268 bp PCR product using CI’ 1; two fragments with a predicted size of I97 and 7 I bp are obtained by electrophoresis

strongly imniunoreactive for both TSP and CD36 (Fig. 3 C and 3 D), while the basement membrane was negative for TSP (Fig. 3 C) and CD5 1 (not shown). Budding, sprouting and ductal mammary epithelial cells were negative for CD5 1 from the 15th to the 20th week of gestation. However, the malpighian epithelium (Fig. 4 A) and preadipocytes of the fat pad precursor tis-

Because of the small amount of fetal breast tissue available, conventional RNA detection methods were not possible. The presence of TSPl transcripts in nascent breast was therefore confirmed by synthesizing and amplifying a double stranded cDNA using PCR assay. A 268 bp PCR fragment which spans nucleotides 904-1 17 1 was obtained (Fig. 5 , lane 2). According to the TSPl nucleotide sequence [25], a Cfo I site was localized at position 71 in the 268 bp PCR fragment, suggesting that digestion of the PCR product with Cfo I could generate two fragments of 197 and 71 bp, respectively. Partial restriction mapping of the 268 bp PCR fragment with Cfo I was therefore performed. Two fragments with a predicted size of 197 and 71 bp, respectively, were obtained (Fig. 5, lane 3), further confirming the presence of TSPl transcripts in nascent breast tissue.

In situ hybridization To further define the source of TSP during development of the human mammary gland, specific localization of TSPl

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Fig. 6. Localization of TSPl mRNA i n mammary bud (A, B) and ducts (C, D) of a 24-week-old female fetus by in situ hybridization. A, C Dark-field photomicrographs of tissue sections demonstrating the silver grains specifically localized over epithelial cells of the

mammary bud and ducts using an antisense probe. B, D Dark-field photomicrographs of adjacent sections showing nonspecific hybridization using a sense probe. Bar, 20 pm

mRNA was performed by in situ hybridization (Fig. 6). With the specific antisense probe there was high-intensity hybridization over epithelial cells of the mammary bud (Fig. 6 A ) . I n addition, a higher density of silver grains seemed to be present at the tip of the mammary bud. Comparison with hybridization using the sense probe indicated minimal hybridization over budding epithelial cells (Fig. 6 B). In fetal mammary ducts, there was high-intensity hybridization over myoepithelial cells (and/or luminal epithelial cells?) (Fig. 6 C). By contrast, minimal hybridization was observed with the sense probe (Fig. 6 D).

is followed by an initial branching of the mammary sprout which forms the ductal tree system of the mammary gland U21. The aim of this study was to investigate the localization of TSP, CD36 and CD5 1 during the different steps of the mammary gland developmental process. Using both in situ hybridization and PCR RNA assay, we have shown that TSP mRNA is expressed by budding, sprouting and ductal mammary epithelial cells. At the protein level, TSP was present in the dense mesenchyme and at the cell surface of mammary epithelial cells when using immunohistochemistry. Moreover, increased TSP deposits were observed at the end bud tip where epithelial cells invade the surrounding mesenchyme. Similarly, during development of the cerebellar cortex of the mouse embryo, granule cells preparing to migrate also exhibit a striking enrichment of TSP, and anti-TSP antibodies inhibit granule cell migration from explant cultures of cerebellar cortex [30]. TSP has anti-adhesive properties [12, 21, 28, 331 and it also stimulates migration of mammary myoepithelial

Discussion During development of the human mammary gland, epithelial cells enlarge and form a bulb-shaped structure: the mammary bud [32]. The mammary bud elongates by rapid cellular proliferation, forming the mammary sprout which then invades the fat pad precursor tissue [32]. This

~-

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cells [37]. Taken together, these findings strongly suggest that highly localized TSP deposits destabilize cell-matrix interactions and promote migration of mammary epithelial cells in the surrounding mesenchyme. Surprisingly, TSP does not codistribute with two of its receptors (CD36 and CD51) [4, 24, 271 in budding and sprouting mammary epithelial cells, while it does with the a2 subunit integrin of the collagen receptor (CDw49b). This indicates that CD36 and CD51 do not serve as TSP receptors when mammary epithelial cells are invading the surrounding mesenchyme. In vitro, a, (CD51) functions as a TSP cellular receptor when it is associated with the p3 subunit integrin [24]. However, the a, subunit can also associate with several different p subunits @, , pl, p,, Po,p 8 ) [6], the p3 subunit does not appear to be expressed in human adult breast [20], and it is not known whether the a, subunit binds TSP when it is associated with the other p subunits. The localization of CD36 in preadipocytes of the fat precursor tissue of nascent breast confirms and extends previous findings obtained with adipocytes of the adult breast tissue [13]. These findings are also in accordance with the recent cloning of a rat adipocyte membrane protein homologous to CD36 [2]. It has been suggested that CD36 could mediate binding and/or transport of long-chain fatty acids in rat preadipocytes [2]. Beside its role as a TSP [4, 271 and a collagen receptor [17], CD36 could also function as a fatty acid acceptor during preadipocyte differentiation in fetal mammary gland. Although CDw49b has also been postulated as a TSP receptor during adhesion of human blood platelets [39], its localization in epithelial cells is most probably related to the presence of type IV collagen in the basement membrane of the mammary bud

WI. As formation of the ductal tree system occurs, TSP becomes localized at the myoepithelial-stromal junction of fetal mammary ducts. However, in lactating mammary ducts of a newborn, TSP disappears from the myoepithelial-stromal junction of ducts, while it becomes selectively localized at the apices of secretory epithelial cells, confirming our previous findings that TSP is present in breast secretions during initiation of lactation in humans [ 151. CD36, which is closely related to mammary epithelial cell surface protein PAS-IV [ 171, is also selectively deposited in secretory epithelial cells. These findings are in agreement with the presence of PAS-IV in breast secretions during lactation [17].The fact that both CD36 and PAS-IV bind TSP in vitro [7] strongly suggests that CD36 is a TSP cell surface receptor in fetal lactating ducts as it has been previously reported for adult lactating ducts 1131. It seems therefore that the distribution of TSP is dependent on the secretory activity of human mammary ducts. I n conclusion, our findings support the existence of an important role for TSP during development of the human fetal mammary gland. Ultimately, variations in the appearance and distribution of TSP and CD36 during the different steps of the mammary gland developmental process may lead to changes in TSP functions, resulting in migration of budding epithelial cells or differentiation of ductal epithelial cells.

Acknowledgements. The technical assistance of P. Peysson is gratefully acknowledged. This work was supported by grants from La FCdCration Nationale des Groupements des Entreprises FranGaises dans la Lutte contre la Cancer (P.C.), the National Institutes of Health, National Heart, Lung and Blood Institute (HL 28749 and HL 42443; J.L.), the Association pour la Recherche sur le Cancer (L.F.) and the Ligue contre le Cancer de la Drome (N.B.). Thanks also to J. Carew for editorial assistance.

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