Cell Tissue Res (1992) 268:167-177

Cell&Tissue Research 9 Springer-Verlag 1992

Changes in the extraeellular matrix of the normal human breast during the menstrual cycle J.E. Ferguson 1, A.M. Schor a, A. Howell t, and M.W.J. Ferguson 2 1 CRC Department of MedicalOncology,Christie Hospital and Holt RadiumInstitute,WilmslowRoad, Withington, Manchester M20 9BX, UK 2 Department of Cell and Structural Biology,Universityof Manchester, Stopford Building,Oxford Road, Manchester M13 9PT, UK Received January 7, 1991 / Accepted December 3, 1991

Summary. The normal human mammary gland undergoes a well defined sequence of histological changes in both epithelial and stromal compartments during the menstrual cycle. Studies in vitro have suggested that the extracellular matrix surrounding the individual cells plays a central role in modulating a wide variety of cellular events, including proliferation, differentiation and gene expression. We therefore investigated the distribution of a number of extracellular matrix molecules in the normal breast during the menstrual cycle. By use of indirect immunofluorescence, with specific antibodies, we demonstrated that laminin, heparan sulphate proteoglycan, type IV collagen, type V collagen, chondroitin sulphate and fibronectin undergo changes in distribution during the menstrual cycle, whereas collagen types I, III, VI and VII remain unchanged. These changes were most marked in the basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts surrounding the ductules where basement membrane markers such as laminin, heparan sulphate proteoglycan, and type IV and V collagens appear greatly reduced during the mid-cycle period (days 8 to 22). These results suggest that some extracellular matrix molecules may act as mediators in the hormonal control of the mammary gland, whereas others may have a predominantly structural role. Key words: Mammary gland Extracellular matrix Menstrual cycle - Breast cancer - Immunohistochemistry - Epithelial cell behaviour - Human

Human mammary ductules are surrounded by a specialised cuff of loose connective tissue, the intralobular stroma. Ductules and intralobular stroma form a functional unit within the lobule. Lobules in turn are surrounded by more dense connective tissue, the interlobular stroma. The intralobular stroma is composed principally of inOffprint requests to :

M.W.J. Ferguson

tralobular fibroblasts, their matrix, and numerous small blood vessels. This stroma undergoes histological and chemical changes during the menstrual cycle suggesting that it is responsive to ovarian steroid hormones. Thus the intralobular stroma appears morphologically homogenous with the interlobular stroma in the first and fourth weeks of the cycle, but acquires a loose foamy appearance in the second and third weeks (Fanger and Ree 1974; Longacre and Barrow 1986). Similarly, an increase in the intralobular concentration of neutral mucopolysaccharides has been documented in the 2nd and 3rd weeks of the cycle and a reduction in acid mucopolysaccharides in the first week of the cycle (Ozello and Speer 1958). We have previously documented that tenascin is cyclically regulated in the human breast (Ferguson et al. 1990). In addition to the stromal changes, cyclical histological events occur in the mammary epithelium which changes from small cuboidal cells in the first half of the cycle (day 1 to 15) to tall, columnar, welldifferentiated cells in the second half of the cycle (day 16-30) (Longacre and Bartow 1986). Periodic epithelial mitotic and apoptotic peaks also occur in relation to cyclical ovarian steroid secretion (Ferguson and Anderson 1981 ; Vogel et al. 1981 ; Potten et al. 1988). The epithelium and the intralobular stroma are separated anatomically by the epithelial-stromal junction which consists of the basement membrane in contact with the basal surfaces of the epithelial and myoepithelial cells, the sub-basement membrane zone containing anchoring fibrils and a monolayer of highly attenuated fibroblasts - the delimiting layer of fibroblasts. Intralobular stromal vessels are frequently found in contact with the delimiting layer of fibroblasts without intervening stroma (Ozello 1970). In contrast to the intralobular stroma, the interlobular stroma is compact and collagenous, less cellular and is presumed to be less responsive to ovarian steroids. The extracellular matrix appears to be important in modulating cell behaviour in the mammary gland; for example Sakakura et al. (1976) have shown that the stroma is the critical factor in determining mammary glandular

168 m o r p h o l o g y . Similarly, the acquisition o f the epithelial characteristics associated with the differentiated state in vitro (such as t - c a s e i n p r o d u c t i o n in response to prolactin) occur only on floating collagen gels or reconstituted basal lamina and n o t on plastic s u b s t r a t u m (Lee et al. 1984; P a r r y et al. 1987). O t h e r p h e n o t y p i c characteristics such as epithelial cell shape, polarity, g r o w t h (Wicha et al. 1979) and responsiveness to h o r m o n e s ( M c G r a t h 1985) are also highly dependent on the s u b s t r a t u m on which the cells are placed. The question then arises whether the a p p a r e n t l y horm o n a l l y regulated epithelial and stromal events in the n o r m a l h u m a n breast which o c c u r during the menstrual cycle, m i g h t be associated with, or mediated by, changes in the extracellular matrix. In order to investigate this hypothesis we studied the a p p e a r a n c e and distribution o f various extracellular matrix molecules in the n o r m a l breast during four phases o f the menstrual cycle. O u r results suggest that h o r m a l l y regulated epithelial and stromal events in vivo are associated with changes in certain extracellular matrix molecules (laminin, h e p a r a n sulphate p r o t e o g l y c a n , type IV a n d type V collagen, fibronectin a n d c h o n d r o i t i n sulphate). We speculate that changes in the c o m p o s i t i o n o f the extracellular matrix during the cycle m a y alter the response o f the adjacent cells to ovarian steroids and g r o w t h factors.

Materials and methods Thirty-one normal mammary biopsies were obtained from the periphery of the breast in women undergoing operation for benign fibroadenoma or reduction mammoplasty. The corresponding age, final histological diagnosis and day of the menstrual cycle were recorded. Patients ages ranged from 16 to 31 years. The histology of the lesions biopsied were fibroadenoma (24 cases), no abnormality (2 cases), lipoma (1 case), lymph node (1 case) and reduction mammoplasty (3 cases). Biopsies were obtained from 8 patients during the first week of the menstrual cycle (days 1-7), 10 patients during the second week (days 8-15), 8 patients in the third week (days 16-22) and 5 patients during the fourth week of the cycle (days 23 30). Two patients were taking the oral contraceptive pill and twenty nine were either not using contraceptive pills or had not taken the pill in the preceding six months. The tissue was rapidly frozen in liquid nitrogen and stored at - 7 0 ~ C for up to one year. Specimens were embedded in OCT, and sectioned at 5-9 gm using a Leitz Cryostat. The sections were mounted on clean glass slides, air dried and fixed in cold acetone for 10 min. They were either used immediately or stored for up to one month at - 2 0 ~ C. In order to exclude epitope masking in cases of negative results the sections received one of the following pre-treatments : (a) 0.1 M acetic acid or 0.5 M acetic acid - both of which cause swelling of collagen fibres and unmask epitopes of minor collagens; (b) sheep testicular hyaluronidase type III 1 rag/50 ml in 1 M sodium acetate buffer adjusted to pH 5 to digest glycosaminoglycans (Von der Mark 1976, 1982). All pre-treatments lasted for 30 min at room temperature.

Table 1. Source and dilution of primary and secondary antibodies used to study extracelluIar matrix of the human breast Extracellular matrix or other molecule

Primary antibody

Source

Dilution

Secondary antibody Source (all FITC conjugated)

Dilution

Laminin

Rabbit antimouse (polyclonal)

Bethesda Research Laboratories, Paisley, UK

1/4~1/80

IgG sheep anti rabbit

Serotec

1/160

Fibronectin

Rabbit anti human (polyclonal)

Pasteur Institute, Paris, France

1/80

IgG sheep anti rabbit

Serotec

1/160

Collagen I

Rabbit anti human (polyclonal) Rabbit anti human Rabbit anti human Goat anti human Rabbit anti bovine LH7 mouse monoclonal

Pasteur Institute

1/25-1/50

lgG sheep anti rabbit

Serotec

1/160

Pasteur Institute Pasteur Institute Sera-Lab, Cambridge, UK S. Ayad (Manchester University) Irene Leigh ICRF (London Hospital Medical College)

1/50 1/80-1/160 1/100 1/100 1/14

IgG sheep IgG sheep IgG sheep IgG sheep IgG sheep

Serotec Serotec Serotec Serotec Serotec

1/160 1/160 1/160 1/160 1/160

Chondroitin sulphate

Mouse monoclonal

ICN Immunobiologicals, Costa Mesa, Calif., USA

1/80-1/100

IgM sheep anti mouse Serotec

1/100

Heparan sulphate

Rabbit anti EHS P. Bretchley, tumour (polyclonal) St. Mary's Hospital, Manchester, UK

1/100

IgG sheep anti rabbit

Serotec

1/160

Vimentin

Mouse monoclonal

ICN Immunobiologicals

1/100

IgG sheep anti mouse Serotec

1/160

Dakopatts, Glostrup, Denmark

l/t00

IgG sheep anti rabbit

1/160

III IV V VI VII

Factor VIIIRabbit anti human related antibody (von Willebrand factor)

anti anti anti anti anti

rabbit rabbit goat rabbit mouse

Serotec

169 The sections were incubated in a humidified chamber with primary antisera as detailed in Table 1 for 1 h and then washed for 3 x 5 rain periods with phosphate-buffered saline (PBS). After a second incubation with fluorescein-conjugated secondary antibody (Table 1) and washing with PBS, sections were mounted in glycerol containing PBS and 1,4 diazobicyclo 2,2,2 octane (DABCO) to prevent fading (Johnston et al. 1982) and coverslipped. Examination for immunofluorescencewas carried out using a Leitz Orthoplan incident epiftuorescent microscope (equipped with a Pleomopak filter). All slides were examined by two observers independently, and the percentage staining in structures known to completely surround the mammary ductules (i.e., basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts) was recorded semi-quantitatively as an estimate (in 10% increments) of the percentage of the circumference of the ductule which was positively stained. A value of 100% represents visible staining of the entire circumference of the structure and a value of 50% means that staining is visible over only half of the structure's circumference. No quantification of the brightness of the immunofluorescent staining was attempted as this is known to vary according to the mean variations in section thickness, affinity of the primary antibodies and dilution of the primary and secondary antibodies. Photographs were taken using Ektachrome ASA 160 colour tungsten

film from which black and white prints were made. Control sections were prepared as above except that PBS was used instead of the primary antisera. Another set of absorption controls utilised primary antibody previously absorbed by incubating with excess quantities of the specific antigen, e.g., type III collagen, to check the specificity of the primary antisera.

Fig. 1 A-D. Definition of various histological regions of human breast using antisera to extracellular matrix molecules. A Type IV collagen immunostaining the basement membrane (B), sub-basement membrane zone (S) and delimiting layer of fibroblasts (DF) of a ductule (D). The thin, innermost fine line adjacent to the epithelium (E) is the basement membrane (B). Punctate granules of type IV collagen are visible in the intralobular stroma (AS). Blood vessels (BV) are shown in both intralobular stroma (AS) and interlobular stroma (ES). Scale bar: 100 gm. B Type VII collagen immunostaining in the sub-basement membrane zone (S).

Brightly staining fibrils (F) traverse its width. None of the other structures including the intralobular stroma (AS), epithelium (E) and lumen shown in the breast stain for type VII collagen. Scale bar: 50 gin. C Type V collagen immunostainingthe delimiting layer of fibroblasts (DF) The sub-basement membrane zone (S) (arrow) and basement membrane (B) are also stained. Scale bar: 100 gm. D Large numbers of fibroblasts stained by antiserum to vimentin in the intralobular stroma (AS) and only a few clusters in the interlobular stroma (ES). Positive staining in small blood vessels in the interlobular stroma is also shown (BV). Scale bar: 100 gm

Results All c o n t r o l sections for all a n t i b o d i e s showed n o staining. N o n e o f the a n t i b o d i e s to the extracellular m a t r i x molecules studied stained the m a m m a r y e p i t h e l i u m or the m y o e p i t h e l i u m . There was n o difference in the distrib u t i o n of the extracellular m a t r i x molecules as a result o f the u n m a s k i n g procedures.

The epithelial-stromal junction The b a s e m e n t m e m b r a n e p r o p e r is seen as a very thin, brightly s t a i n i n g line a d j a c e n t to the basal surfaces o f

170 the myoepithelial cells, surrounding each ductule. It is visualised by staining with antisera to type IV collage n (Fig. I a). The sub-basement membrane zone appears as a broad band of fluorescence immediately below the basement membrane and contains brightly stained fibrils perpendicular to the basement membrane which traverse its width. This zone is best visualised with type VII collagen antibody (Fig. I b) which localises exclusively in the sub-basement membrane zone and stains anchoring filaments from the basement membrane. The delimiting layer of fibroblasts appears as an incomplete thin layer of fibroblasts surrounding and adjacent to the sub-basement membrane zone and is shown stained with antisera to type V collagen in Fig. 1 c. The intralobular stroma surrounding the ductules contains large numbers of fibroblasts (demonstrated in Fig. 1 d by staining with vimentin antisera). Type 1V collagen (Fig. I a), type V collagen (Fig. 1 c), heparan sulphate proteoglycan (Fig. 2a), laminin (Fig. 2c) and fibronectin (Fig. 2d) were all present in the basement membrane, sub-basement membrane zone and delimiting layer o f fibroblasts surrounding the ductules. These

three layers showed similar intensity staining for the different molecules mentioned above, with the possible exception of type V collagen which appeared to stain the basement membrane and the delimiting layer of fibroblasts more intensely than the sub-basement membrane zone. Small ductule like structures (Fig. 2a) composed of epithelial cells but without a lumen were seen adjacent to normal ductules in some lobules. The entire surface of such structures stains homogeneously with antibodies to laminin, heparan sulphate proteoglycan and type IV and V collagens, but they have no distinguishable basement membrane or deliminating layer of fibroblasts. Some of these structures may be oblique sections of small branching ductules with a particularly broad subbasement membrane zone and others may be branching ductular endbuds cut en face through their basal surfaces, as epithelial outlines are often visualised through the staining (e.g., Fig. 2c).

Fig. 2. A Small ductule-like structures (DL) without lumina, are shown stained with antiserum to heparan sulphate proteoglycan. They are easily distinguishable from normal ductules (D) and blood vessels (BI/). ES Interlobutar stroma; F fibroblasts. Scale bar: 100 gin. B Immunostaining of Factor VIII-related antigen in small blood vessels in a large lobule. Large numbers of vessels are found in the intralobular stroma (AS) and surrounding the ductutes (D) and very few in the interlobular stroma (ES). Scale bar." 200 gin.

C Immunostaining of laminin in the basement membrane (B), subbasement membrane zone (S) and delimiting layer of fibroblasts (DF) surrounding ductules (D). The interlobular stroma (AS) and blood vessels (BV) also stain. Scale bar: 100 gin. D Fibronectin immunostaining the delimiting layer of fibroblasts (DF) (arrow) adjacent to the sub-basement membrane zone (S) of a small ductule (D). A second cuff of fibroblasts (F) surrounds the ductules. Scale bar: 50 ~tm

171

The intralobular stroma Small vessels, visualised by staining with factor VIII related antigen are very numerous in the intralobular stroma and sparse in the interlobular stroma (Fig. 2 b). The basement membrane of the small vessels of the intralobular stroma stained with collagens type IV (Fig. 1 a), type V (Fig. 1 c), heparan sulphate proteoglycan (Fig. 2a), laminin (Fig. 2c) and fibronectin (Fig. 2d). Each of those molecules, with the exception of fibronectin, was also present in the intralobular stroma as fine streaks and punctate deposits(Figs, l a, c; 2a, c). Fibronectin (Fig. 2d) [and chondroitin sulphate (Fig. 3c)] was present as a fine reticular network often enveloping ductules and vessels in the intralobular stroma.

The interlobular stroma The interlobular stroma contained relatively few fibroblasts arranged in small clusters (Fig. 3 a). Heparan sulphate proteoglycan and collagens type IV and V were present as fine streaks on some of these clusters. By contrast to these molecules, fibronectin (Fig. 3b) and

Fig. 3. A Vimentin staining clusters of fibroblasts in the interlobular stroma (ES). Small vessels (BV) are also shown. Scale bar: 100 ~tm. B A fine reticular network of fibronectin envelops the ductules (D) and blood vessels (BV) in the intralobular stroma (AS). This contrasts with the linear streaks of fibronectin in the interlobular stroma (ES) and are particularly numerous adjacent to the lobule.

chondroitin sulphate (Fig. 3 c) were more abundant in the interlobular stroma than the intralobular stroma. In each case, fine fibrils were visible on the surface of fibroblast clusters and there were long parallel bands in the stroma which increased in number and became almost concentric adjacent to lobules (Fig. 3b). Small vessels were sparse, and large vessels seen infrequently. When present, large vessels had fine streak of heparan sulphate proteoglycan (Fig. 3d), chondroitin sulphate and fibronectin in both media and adventitia, in addition to the component molecules of the basement membrane. Fat cells were seen occasionally in the interlobular stroma. The basement membrane of these cells contained heparan sulphate proteoglycan, fibronectin, type IV and type V collagens and laminin in a thin continuous line (not shown).

Cyclical changes in the distribution of laminin, type I V collagen, type V collagen, heparan sulphate proteoglycan, chondroitin sulphate and fibronectin during the menstrual cycle Laminin, heparan sulphate proteoglycan, collagens type IV and V, fibronectin and chondroitin sulphate under-

Scale bar: 100 gm. C Chondroitin sulphate staining in a reticular network around ductules (D) in the intralobular stroma (AS). Compare with B. Scale bar: 100 p.m. D Heparan sulphate proteoglycan in the basement membrane (B), media (M) and adventitia (A) of a large venule and arteriole in the interlobular stroma. E Endothelium. Scale bar: 100 gm

172

Fig. 4A-D. Immunostaining of type IV collagen during weeks 1 to 4 of the menstrual cycle. In week 1 (A) the basement membrane (B), sub-basement membrane zone (S) and delimiting layer of fibroblasts (DF) are continuous. Punctate deposits of type IV collagen are seen in the intralobular stroma (AS) and linear streaks of type IV collagen in the interlobular stroma (ES). In week 2 (B) and week 3 (C) there are large areas of discontinuity of staining in the basement membrane (B), sub-basement membrane zone (S)

and delimiting layer of fibroblast (DF). In week 4 (D) the three layers again appear continuous. Punctate deposits of type IV collagen in the intralobular stroma (AS) are also reduced in weeks 2 (B) and 3 (C) in comparison with weeks 1 (A) and 4 (D). Note that staining in the blood vessels (BI/) of the intralobular stroma (AS) in unchanged during the cycle. D Ductule; F fibroblasts. Scale bar: 100 gm

went marked changes in appearance and distribution during the menstrual cycle (Table 2). The most dramatic changes were seen in the epithelial stromal junction where similar cycle-related changes occurred in the distribution of type IV collagen (Fig. 4 a-d), type V collagen (not shown), heparan sulphate proteoglycan (not shown) and laminin (Fig. 5a-d). Antibodies to each of these molecules stained 7 5 % - 1 0 0 % of the circumference o f the basement membrane and sub-basement membrane zone and approximately 5 0 % - 7 5 % of the delimiting layer o f fibroblasts in week 1 (days 1-7) (e.g., Fig. 4a) and week 4 (days 21-28) (Fig. 4d) of the menstrual cycle. A consistent and marked reduction occurred in the 2nd and 3rd weeks of the cycle with antibodies to type IV collagen, laminin (Figs. 4 b, c; 5 b, c, respectively), heparan sulphate proteoglycan and type V collagen staining 1 0 % - 5 0 % o f the basement membrane circumference. 10% to 25% of the sub-basement membrane zone o f large ductules stained with antibodies to heparan sulphate proteoglycan, type IV collagen and type V collagen but only 0-10% of the sub-basement membrane zone o f small ductules stained during the 2nd and 3rd

weeks of the cycle. In all weeks of the cycle, laminin stained in a continuous line in the sub-basement membrane zone, but staining became paler and narrower in weeks 2 and 3 (Fig. 5 b, c). Staining of the delimiting layer of fibroblasts was dramatically reduced. In the 2nd week of the cycle, type V collagen stained only 1 0 % 25% of the circumference of this layer. In the 3rd week of the cycle, laminin, type IV collagen, heparan sulphate proteoglycan and type V collagen all stained but only covered 1 0 % - 5 0 % of the circumference. Noticeable, but lesser, changes occurred in the intralobular stroma with a reduction in the number and intensity o f granular and streaking stained with type IV collagen, type V collagen, laminin and heparan sulphate proteoglycan in weeks 2 and 3 by comparison to weeks 1 and 4 (Figs. 4a, b; 5a, b). The pattern of staining observed with the chondroitin sulphate antisera was similar to that of heparan sulphate proteoglycan, laminin etc. described above with the exception that chondroitin sulphate was not present in the basement membrane. Increased staining covering 7 5 % 100% of the delimiting layer and sub-basement mere-

173

Fig. 5A-D. Laminin immunostaining during the four weeks of the menstrual cycle. A Week 1. The basement membrane (B), sub-basement membrane zone (S) and delimiting layer of fibroblasts (DF) are continuous and brightly stained. B, C Weeks 2 and 3. Reduced intensity and patchy staining of the basement membrane (B), sub-

basement membrane zone (S) and delimiting layer of fibroblasts (DF). D Week 4. All three layers stain brightly and are continuous. There is no alteration in the pattern of blood vessel staining throughout the cycle. AS Intralobular stroma; ES interlobular stroma. Scale bar: 100 gm

brane circumferences was observed in weeks 1 and 4 of the cycle and a reduced staining in these two layers in weeks 2 and 3. A minor reduction in the intralobular staining was also seen in the second and third weeks. Changes observed in fibronectin staining during the menstrual cycle were less well-defined. There was greater overlap in ductular appearance between adjacent weeks of the cycle, than for other extracellular matrix molecules studied. Changes in the delimiting layer of fibroblasts and interlobular stroma mirrored those of laminin and heparan sulphate proteoglycan with reduced staining in the second and third weeks of the cycle. These changes were asynchronous with those in the sub-basement membrane zone in which most staining was observed in weeks 2 and 3 ( 5 0 % - 7 5 % of the circumference) and least in weeks 1 and 4 (0-10% and 2 5 % - 7 5 % of the circumference, respectively). Fibronectin in the basement membrane was continuous throughout the cycle. The structures tentatively described as endbuds (Fig. 2 c) were particularly evident in the third and fourth weeks of the cycle and were rarely seen in the first and second weeks. In general quantitative changes in the extracellular matrix during the cycle were most pronounced in the smaller ducts and ductules of the lobules, whereas major ducts showed least changes. It should

be emphasised that in spite of these compositional changes the basement membrane has been shown previously to remain histologically intact throughout the cycle as assessed by transmission electron microscopy (Fanger and Ree 1974). There were no changes in the distribution or appearance of collagen types I, III, VI and VII during the menstrual cycle. Similarly, no extracellular matrix changes were observed in the interlobular stroma (see Table 3). The overall histological appearance of the lobules was remarkably constant for any given time of the cycle, and no changes were seen in the basement membranes of fat cells or blood vessels (Figs. 4 a d; 5 a-d). The distribution of molecules which do not change in the menstrual cycle in the normal breast, i.e., types I, Ill, VI and VII collagens Type I and III collagens were present as fine fibrils and were similarly distributed. They were found predominantly in the interlobular stroma, where fibrils were closely grouped immediately adjacent to lobules and widely separated in the intervening stroma. Fewer and sparser streaks were present in the intralobular stroma and on the delimiting layer of fibroblasts (not shown).

174 Table 2. Changes in localisation of staining in the human breast with antisera to certain extracellular matrix molecules during weeks 1, 2, 3 and 4 of the menstrual cycle. - , Absent (no staining) ; +, 10%-25%; 2 + , 25%-50%; 3+, 50%-75%; 4 + , 75%-100% positive staining in observed zones. CIV, Type IV collagen; HSPG, heparan sulphate proteoglycan; LN, laminin; CV, type V collagen; FN, fibronectin; CS, chondroitin sulphate. Sample number for each week is 4~6

Location

ExtraMenstrual cycle (week) cellular matrix 1 2 3 molecule

Basement membrane

CIV HSPG LN CV FN CS Sub-basement CIV membrane HSPG zone LN CV FN CS Delimiting CIV layer HSPG of fibroblasts LN CV FN CS Intralobular CIV stroma HSPG LN CV FN CS Interlobular CIV stroma HSPG LN CV FN CS

4

4+ 4+ 4+ 4+/3+ 4+

+/2+ + 2+/3+ 2+ 4+

2+/+ 2+/3+ 2+/3+ 2+ 4+

4+ 4+ 4+/3+ 4+ 4+

4+ 4+ 4+ 4+

2+/3+ 0-->2+ 4 + Pale +/-

2+ 4+ 3+ 4+ 4+ Pale 4+ + 2+/3+

+/-

3+

3+

2+/3+

4+

+/2+

+/2+

2+/3+

3+ 2/3+ 3+ 4+ 3+ 4+ + 2+ + 2+ 3+ 3+

+ 2+ 4/3+ + + + + + +

+ +/2+ + 3+ 2+ 4+/3+ + + + + 2+ +

2+ 3+ 3+ 4+ 2+/3+ 4+ + 2+ + 2+ 3+ 3+

+

--

__

--

+

+/-

+

+

+/2+ 2+

-

+

2+

2+ +

2+ +

2+ +

Type VI collagen was u b i q u i t o u s in the n o r m a l breast (Fig. 6 a, b). It stained the s u b - b a s e m e n t m e m b r a n e zone as a b r o a d a n d c o n t i n u o u s b a n d , separated f r o m the i n t r a l o b u l a r s t r o m a b y a clear gap. T h e i n t r a l o b u l a r s t r o m a c o n t a i n e d b r o a d b a n d s o f type VI collagen in a n e t w o r k w h i c h e n v e l o p e d the ductules u p to the n o n s t a i n i n g gap b e t w e e n the i n t r a l o b u l a r s t r o m a a n d the s u b - b a s e m e n t m e m b r a n e zone. The i n t e r l o b u l a r s t r o m a also c o n t a i n e d b r o a d b a n d s o f type VI collagen which

Fig. 6. A Type VI collagen immunostainingin the epithelial stromal junction, intralobular stroma (AS) and interlobular stroma (ES) in the normal breast. Scale bar: 100 gin. B Higher magnification of A showing immunostainingfor type VI collagen in the basement membrane (B), sub-basement membrane zone (S) and delimiting layer of fibroblasts (DF) surrounding ductule (D). Scale bar: 50 gm

were m o r e n u m e r o u s a n d a r r a n g e d in whorls close to the l o b u l a r surface (Fig. 6a). The s t r o m a between these b a n d s was pale a n d stained h o m o g e n e o u s l y . T h e d i s t r i b u t i o n o f type VII collagen was restricted to the s u b - b a s e m e n t m e m b r a n e zone in which it appeared as a b r o a d layer o f fine fibrils completely surr o u n d i n g the ductules (Fig. 1 b). C o l l a g e n types I, III, VI a n d VII were n o t detected in the epithelium, b l o o d vessels a n d b a s e m e n t m e m b r a n e s of fat cells.

Discussion

Cyclical variation in mammary epithelial proliferative activity (Ferguson and Anderson 1981), histological ap-

Table 3. The distribution in the human breast of extracellular matrix molecules which do not change during the menstrual cycle

ECM molecule

Basement membrane

Sub-basement membrane zone

Delimiting layer of fibroblasts

Intralobular stroma

Interlobular stroma

Vessels

Type I collagen Type III collagen Type VI collagen Type VII collagen

3+ -

3+ 4+

+ + 3+ .

2+ + 3+ .

3+ 2+ 3+

+ large vessels only -

.

.

175 pearance (Longacre and Bartow 1986), and oestrogen and progesterone receptor content have been documented (Silva et al. 1983; Pollow et al. 1977). This study documents cyclical changes in the extracellular matrix molecule profile of the human breast. Two major compartments, i.e., the epithelial-stromal junction and the intralobular stroma underwent changes in the extracellular matrix molecules during the cycle. The most dramatic changes occurred in the basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts which we found to contain variable quantities of laminin, type IV collagen, heparan sulphate proteoglycan and type V collagen depending on the time of the menstrual cycle. A general pattern of cyclical changes emerged whereby maximal quantities of these molecules were present in weeks 1 and 4 of the cycle, and reduced staining occurred in weeks 2 and 3, implying possible co-regulation of expression. Other extracellular matrix molecules for example, tenascin (Ferguson et al. 1990) and fibronectin in the sub-basement membrane zone, were maximally expressed in the 4th and 3rd weeks, respectively, with lesser amounts in weeks I and 2 suggesting that a different regulatory system was operable, or that different patterns of extracellular matrix molecules were produced in response to the same stimulus. These changes we observed are not likely to be artefactual due to masking of epitopes or different isoforms of the molecule, as enzymic pretreatments to digest glycosaminoglycans or to expose collagen epitopes did not change the staining patterns observed. Additionally all primary antisera were polyclonal and presumably recognised numerous epitopes on the molecules. Furthermore, the basement membrane of blood vessels, although containing the same extracellular matrix molecules as the ductular basement membrane showed no changes during the cycle. This suggests that the varia-" tions observed in the ductules during the menstrual cycle reflect hormonally induced changes in extracellular matrix synthesis or degradation or a combination of both. Surprisingly, the delimiting layer of fibroblasts and sub-basement membrane zone expressed extracellular matrix molecules which are normally considered exclusive to the basement membrane proper, such as type IV collagen, laminin and heparan sulphate proteoglycan. Others, such as collagens type V and VI and fibronectin were also present in all three layers, i.e. basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts, whereas chondroitin sulphate was found only in the latter two layers (and not in the basement membrane) and type VII collagen was found exclusely in the sub-basement membrane zone. Differences were observed between the inter- and intralobular stroma regarding the composition and distribution of the extracellular matrix. Fine granular extrabasement membrane deposits of laminin, type IV collagen, type V collagen and heparan sulphate proteoglycan were found in the intralobular stroma, and of these only heparan sulphate proteoglycan and type IV and V collagens were also present in the interlobular stroma. Conversely, type I and III collagen were abundant in

the interlobular stroma and very sparse in the intralobular stroma. Thus, there is some differential expression of extracellular matrix molecules between the interlobular and intralobular fibroblasts. Our findings of cyclic modulation of extracellular matrix components in vivo are supported by the in vitro experiments of others. For example, the synthesis of some extracellular matrix molecules (Collagen types I, III, IV, laminin and fibronectin) by normal mouse mammary epithelium is modulated as a function of hormonal status (Park and Bissell 1988). Furthermore, levels of the 62 kD extracellular matrix receptor for laminin are increased by progestins and by estrogen and progestins together in a mammary epithelial cell line (Castronovo et al. 1989). This suggests that variation in synthesis of both extracellular matrix molecules and their receptors in the normal breast may occur as a result of hormonal changes during the menstrual cycle. Precisely how this cyclical regulation of extracellular matrix accumulation in the breast is regulated is unknown. It may be the result of differential synthesis, differential degradation (proteolytic enzymes for matrix degradation are specifically regulated during mouse breast development; Talhouk et al. 1991) or both. Similar effects on the synthesis of matrix macromolecules, proteolytic enzymes or enzyme inhibitors may be mediated secondarily by hormone induced effects on the activity of growth factors in the breast. Studies in vitro have demonstrated that the extracellular matrix modulates cell behaviour. If similar mechanisms are operative in vivo we can speculate that the extracellular matrix during the menstrual cycle may have significant biological effects on cell proliferation, shape, adhesion, differentiation, secretion and migratory activity (Williams and Daniel 1983; Kefalides et al. 1979; Li et al. 1987; Parry et al. 1987; Park and Bissell 1988; Liotta et al. 1979; Wicha et al. 1979, 1980; Emerman et al. 1977; Shannon and Pitelka 1981 ; Suard et al. 1983; McGrath et al. 1985). For example, the reduced quantities of laminin, heparan sulphate proteoglycan, type IV and type V collagen in weeks two and three of the cycle may account for the accompanying low mitotic activity, whereas in week four the replete basement membrane extracellular matrix may exert an increased mitotic stimulus resulting in the peak mitotic activity around day 25_+2 (Ferguson and Anderson 1981). Furthermore, as epithelial exposure to type IV collagen enhances the mitogenic response to EGF (Salomon et al. 198/) periodic removal of this component of the basement membrane may render cells refractory to growth factors in the second and third weeks of the cycle. The extracellular matrix profile appears to be under hormonal regulation in the normal breast. It would be of interest to discover whether alterations in certain extracellular matrix molecules or loss of hormonal responsiveness may be implicated in the pathogenesis of breast disease. If such change do occur, manipulation of the extracellular matrix may provide an additional therapeutic option. The existence of apparently hormonally-regulated changes in the extracellular matrix has important impli-

176 c a t i o n s for t u m o u r p a t h o l o g i s t s utilising i m m u n o f l u o rescent a n t i b o d i e s to b a s e m e n t m e m b r a n e c o m p o n e n t s ( l a m i n i n a n d t y p e IV c o l l a g e n ) to d i s t i n g u i s h b e t w e e n the i n t a c t b a s e m e n t m e m b r a n e o f c a r c i n o m a in situ a n d the i n t e r r u p t e d b a s e m e n t m e m b r a n e o f m i c r o i n v a s i v e disease. A s this s t u d y h a s d e m o n s t r a t e d a m a j o r r e d u c tion, a n d even absence, o f s o m e b a s e m e n t m e m b r a n e c o m p o n e n t s in weeks t w o a n d t h r e e o f the n o r m a l m e n strual cycle, e r r o r s in d i s t i n g u i s h i n g b e t w e e n c a r c i n o m a in situ a n d m i c r o i n v a s i v e d i s e a s e m a y o c c u r a t this time in the cycle When there a r e n a t u r a l l y low levels o f extracellular m a t r i x present. A l t h o u g h we d o n o t k n o w if such r e g u l a t i o n exists in c a r c i n o m a in situ, e r r o r c o u l d be m i n i m i s e d b y c o n f i n i n g b i o p s i e s to weeks one a n d f o u r o f the cycle w h e n the e x t r a c e l l u l a r m a t r i x c o m p o nents o f the n o r m a l b a s e m e n t m e m b r a n e are m a x i m a l l y expressed.

Acknowledgements. The assistance of Mr. Rob Watson of Withington Hospital in obtaining breast samples and the secretarial assistance of Marj0rie Evans is gratefully acknowledged.

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