A second look at methanol.

The a6/,B4 integrin complex has been shown to be expressed in murine tissues at the basolateral aspect of most epithelial cells including the mammary epithelium, thus suggesting that this heterodimer may interact with components of the basement membrane. Because transformation of mammary epithelium frequently results in disappearance of basement membranes and loss of cell polarisation we have analysed in the present study whether expression of the a6/P4 complex is altered in human breast tumours. The results of the present study confirm that in human mammary gland a6 and P4 subunits colocalise at the basolateral aspect of the epithelium. While in benign breast lesions this distribution pattern remains mostly unchanged, in primary carcinomas the expression of both chains is either redistributed over the cell surface or significantly reduced. This altered pattern of expression is paralleled by the lack of detection of basement membrane laminin and collagen type IV. In metastatic lesions the expression of the heterodimer is maintained in most of the lymphonodal foci, but less frequently detected in metastasis localised in the pleural cavity and in parenchymal tissues. These findings indicate that in breast epithelium expression of the x6/P4 heterodimer is modulated by the presence of basement membrane and is possibly influenced by microenvironmental factors as suggested by the different pattern of x6/P4 expression in nodal and extranodal metastatic foci. Integrins represent an expanding family of heterodimeric receptors (Hynes, 1987) involved in cell-to-cell and cell matrix interactions (Albelda & Buck, 1990). Accumulating experimental evidence points to a major functional role of integrins in the regulation of cell polarity (Fath et al., 1989) and migration (Hemler, 1990) as well as in morphogenesis (Korhonen et al., 1990). It has also been proposed that derangement of integrin expression may be responsible for a number of aberrant cell behaviours during tumour onset, which is formed by the non covalent association of a6 and P1 chains is of particular interest since it represents a non promiscuous receptor for the basement membrane glyco-protein laminin (Sonnenberg et al., 1988). However, the a6 chain can alternatively associate with a different chain to form the a6/,B4 heterodimer (Sonnenberg et al., 1988a; Hemler et al., 1989; Kajiji et al., 1989) whose receptorial activity is not yet fully characterised. Detailed immunohistochemical studies of murine tissues (Sonnenberg et al., 1990) have revealed that a6, P4 and P1 codistribute in most epithelia including the mammary epithelium at the basolateral aspect, thus suggesting that …

Integrins represent an expanding family of heterodimeric receptors (Hynes, 1987) involved in cell-to-cell and cell matrix interactions . Accumulating experimental evidence points to a major functional role of integrins in the regulation of cell polarity (Fath et al., 1989) and migration (Hemler, 1990) as well as in morphogenesis (Korhonen et al., 1990). It has also been proposed that derangement of integrin expression may be responsible for a number of aberrant cell behaviours during tumour onset, progression and metastatic spreading (Plantefaber & Hynes, 1989;Ruoslahti & Giancotti, 1989;Dedhar & Saulnier, 1990;Giancotti & Ruoslahti, 1990).
In this context the VLA6 (Sonnenberg et al., 1987) integrin which is formed by the non covalent association of a6 and P1 chains is of particular interest since it represents a non promiscuous receptor for the basement membrane glycoprotein laminin (Sonnenberg et al., 1988). However, the a6 chain can alternatively associate with a different chain to form the a6/,B4 heterodimer (Sonnenberg et al., 1988a;Hemler et al., 1989;Kajiji et al., 1989) whose receptorial activity is not yet fully characterised. Detailed immunohistochemical studies of murine tissues (Sonnenberg et al., 1990) have revealed that a6, P4 and P1 codistribute in most epithelia including the mammary epithelium at the basolateral aspect, thus suggesting that o6/P4 dimers physically interact with some basement membrane component/s which may in turn modulate this expression and cellular compartimentalisation (Fath et al., 1989). The observation that transformation of mammary epithelium is frequently associated with lack of basement membranes (Ozzello, 1979;Natali et al., 1984;Birembaut et al., 1985;Tsubura et al., 1988) provides the opportunity to test this hypothesis through the comparative analysis of o6/P4 expression in normal and transformed primary and metastatic human mammary epithelium. We report here that in human breast tumours the lack of laminin and collagen type IV i.e. basement membranes is associated with a significantly reduced expression of oc6/P4 as well as loss of its polarised pattern of expression.

Tissues
Surgical biopsies of normal, benign and malignant tumour tissues were collected following ablative surgery from patients free of chemo and radiotherapy. Tissues were snap frozen in liquid nitrogen. From each specimens consecutive 4 cryostat sections were obtained which were fixed in cold absolute acetone for 10 min. Fixed sections were either immediately used in immunohistochemical assay or kept frozen at -3OC with no loss of serological activity. Fixed sections stained with 1% toluidine blue were used to evaluate the histological features of the tissues.
Immunohistochemical assay Indirect immunoperoxidase (IIP) staining was performed by employing on consecutive sections of the same specimen primary MoAbs (25 to 50 fig ml-') and a commercially available avidin-biotin staining kits (Vector Lab., Burlingame, Ca., USA). Because the affinity of MoAbs was unknown the incubation with tissue sections was prolonged for 18 h. Negative controls consisted of tissue sections incubated with irrelevant MoAb. The positive stain of the vascular walls observable with antibodies provided a positive control in each specimen studied. The immunoenzymatic reaction employed 3-amino-9-ethylcarbazole as a chromogenic substrate and Mayer's hematoxylin as nuclear counterstain followed by mounting in buffered glycerol. Indirect immunofluorescence was done as described (Natali et al., 1981)

Results
Expression of c6 and 134 subunits in normal mammary epithelium and benign breast lesions Immunohistochemical analysis of normal breast tissue revealed a consistently strong stain for a6 and P4 which outlined the outer aspect of acini and ducts independently from the discontinuous (acini) and continuous (ducts) disribution of myoepithelial cells. A heterogenous stain of the lateral aspect of luminal cells was seen with MoAb 135.13C to x6 and was, even more pronounced with antibodies to P4 chain. Staining of a6 and P4 at the basal aspect of luminal cells was rarely seen. By indirect immunofluorescence which in our hands allowed a higher resolution, an ordered punctuate stain could be observed for a6 and P4 in section planes running tangential to the basal portion of the ductal and acinar epithelium (Figure la inset). The extent to which myoepithelial and luminal cells contributed to this pattern could not be firmly established. The staining patterns described above were maintained in three types of benign breast tumours tested (Table I). Only in two cases of gynecomastia was the plasma-membrane stain for a6 not associated with detectable levels of P4.
Changes in distribution of a6 and P4 subunits in primary and metastatic breast tumours Evaluation of primary breast tumours of the most common histotypes (Table I) indicates that the expression of x6 and P4 subunits undergoes a number of changes. As a general rule, P4 was never expressed in absence of x6. Three major staining patterns were observed. Staining for az6 and P4 in a significant number of tumours was undetectable at the level of the cell membrane ( Figure lb). This was more frequently seen in lobular and infiltrating ductal carcinomas while it was less common in tubular tumours. Moreover polarised stain for both subunits at the periphery of tumour cell nests (Figure 1c) was rare in most tumour histotypes. The punctuate stain at the base of tumour cell nests was never observed.
The results of the comparative immunohistochemical evaluation of primary tumours and autologous metastasis as well as of metastasis from various anatomical sites are summarised in Table II. Also in this instance three major staining patterns could be observed since staining for P4 was never observed in the absence of detectable a6 chain. Among metastatic lesions, especially those located in lymph nodes (40%) displayed stain for both subunits on tumour cell membrane ( Figure ld). In only four out of 26 metastases was a polarisation of the stain for both chains seen at the periphery of tumour cell nests. While the distribution of both subunits in primary lesions was often (67%) different from that observed in metastatic foci, the distribution of both chains was rather consistent among multiple concomitant autologous metastases. In one case (patient Br) whilst the primary tumour lacked a6 and P4 stain, both chains were expressed in the lymphonodal autologous lesions. As opposed to nodal lesions, parenchymal and particularly pleural metastasis (ten a c 'i   (7) 7  7 Gynecomastia (5)   cases not shown) were found to be negative for a6 and P4 stain over a wide range of MoAb concentrations. Stain for P1 subunit performed in four of these lesions was however consistently positive. All the described staining patterns remained unchanged when using additional MoAb HP2/1 and 3E1 to the a6 and P4 chains respectively.

Relationship between integrin phenotype and basement membrane antigens in primary breast tumours
In order to assess whether the changes in expression and cellular compartmentalisation of the a6 and P4 subunits observed in primary mammary tumours might be associated with an altered distribution of basement membrane, in a selected number of tumours staining of a6 and P4 subunits was compared with the distribution of basement membrane glycoprotein laminin and of collagen type IV. Because a6 chain can alternately dimerise with the P1 subunit to form a non promiscuous receptor for laminin, the expression of this chain was also evaluated in the same specimens. From the results of this study, which are summarised in Table III, the following information could be obtained. On the tumour cell plasma membrane a6 was almost invariably coexpressed with P4 and P11. Polarisation of the stain at the basal aspect of the cells located at the periphery of tumour nests was seen for a6, P4 and P1 and for a6 and P1 only in those tumours which were also stained by the anti-laminin and collagen type IV antiserum. i.e. tumours possessing an antigenically integer basement membrane. Lack of detectable laminin and collagen type IV in five out of seven cases was associated with negative stain for all of the three integrin subunits.

Discussion
The study of the interaction of cells with extracellular matrix components is instrumental in understanding cell differentiation, tissue morphogenesis and the pathogenetic pathways of tumour growth and metastatic spreading. These areas of study are being increasingly explored since the identification of the superfamily of the integrin molecules which mediate a number of specific ligand-receptor interactions between cells and their surrounding milieu (Hynes, 1987;. Different molecular mechanisms may perturb integrin functions during tumour progression, including qualitative and quantitative changes in integrin expression (Hirst et al., 1986;Plantefaber & Hynes, 1989) as well as loss of integrin ligands, i.e. extracellular matrix components (Ruoslahti & Giancotti, 1989;Giancotti & Ruoslahti, 1990). Indeed recent immunohistochemical studies have extended to human tumours the earlier observations obtained either in tissue culture systems or in animal     (McGregor et al., 1989;Wolf et al., 1990;Natali et al., 1991). In agreement with others (Koukoulis et al.,1991;Streuli et al., 1991) we have shown that a6 and 134 integrin subunits are expressed by normal mammary epithelium. This pattern is retained in benign breast tumours whereas it undergoes quantitative and qualitative changes upon malignant transformation. To gain further insights into the possible role of these integrins in tumour progression, we have extended the immunohistochemical analysis to metastatic lesions. This included the evaluation of the two subunits both in primary tumours and multiple concomitant autologous metastases, as well as in metastases sampled from different anatomical sites. Because ultrastructural and immunohistochemical studies have demonstrated the frequent loss of basement membrane in breast carcinomas (Ozzello, 1979;Natali et al., 1984;Birombaut et al., 1985;Tsubura et al., 1988) we have additionally studied whether changes in integrin profile are paralleled by modification of the basement membrane-associated glycoprotein, laminin, and of collagen type IV. In mammary tumours of most common histotypes we have observed a number of modifications in a6 and P4 distribution pattern. Because of the lack of myoepithelial differentiation in the majority of breast tumours (Gould et al., 1980), staining of a6 and P4 pertaining to these non parenchymal cells was rarely seen. The two subunits were mostly undetectable on tumour cells or redistributed over their plasma membrane. These changes, which in our specimens are not related to a given tumour histotype, are almost invariably associated with lack of laminin and collagen type IV at the periphery of the tumour cell nests. Thus the availability of specific ligand/s in the basement membrane appears to direct a polarised expression of the a6/134 heterodimer in normal epithelium, whereas in breast tumour cells the lack of physical interaction between the a6/P4 dimer and the basement membrane may be responsible for some of the above described changes.
In view of the finding that laminin may function as a stop signal to cell migration (Coopman et al., 1991), the transformation-associated changes both in integrin repertoire and basement membrane components may be relevant in determining the invasive behaviour of breast tumour cells.
In contrast to the results reported by Falcioni et al. (1986) and Wolf et al. (1990), the present findings and those of Gould et al. (1991) indicate that tumour progression in breast cancer is not associated with increased levels of expression of a6/134. Our comparative study of primary tumours and autologous metastases has shown a high degree of heterogeneity in expression of the two subunits. This includes differences between the primary neoplasia and autologous metastases (67% of the cases) and to a minor extent among the latter lesions. Thus the modulation of the a6/134 complex does not appear to be related to the metastatic process in breast carcinoma. Nevertheless differences in integrin phenotype between lymph node and parenchymal metastases suggest that expression of the x6/P4 complex may be modulated by local factors such as cytokines (Heino et al., 1989) in addition to extracellular matrix components.
In conclusion our data show that loss of basement membrane components parallels quantitative and qualitative changes in the expression of x6/134 and a6/P1I heterodimers in breast cancer. This may be a crucial step in enhancing local invasiveness of tumour cells, thus facilitating tumour spreading and biological malignancy. This work has been supported by PF ACRO, by the Italian Ministry of Public Health and by Associazione Italiana per la Ricerca sul Cancro. The technical help of Miss Cristina Valentini and the secretarial assistance of Miss Maria Vincenza Sarcone are gratefully acknowledged.