Invasion in breast lesions: the role of the epithelial–stroma barrier

Despite the significant biological, behavioural and management differences between ductal carcinoma in situ (DCIS) and invasive carcinoma of the breast, they share many morphological and molecular similarities. Differentiation of these two different lesions in breast pathological diagnosis is based typically on the presence of an intact barrier between the malignant epithelial cells and stroma; namely, the myoepithelial cell (MEC) layer and surrounding basement membrane (BM). Despite being robust diagnostic criteria, the identification of MECs and BM to differentiate in‐situ from invasive carcinoma is not always straightforward. The MEC layer around DCIS may be interrupted and/or show an altered immunoprofile. MECs may be absent in some benign locally infiltrative lesions such as microglandular adenosis and infiltrating epitheliosis, and occasionally in non‐infiltrative conditions such as apocrine lesions, and in these contexts this does not denote malignancy or invasive disease with metastatic potential. MECs may also be absent around some malignant lesions such as some forms of papillary carcinoma, yet these behave in an indolent fashion akin to some DCIS. In Paget's disease, malignant mammary epithelial cells extend anteriorly from the ducts to infiltrate the epidermis of the nipple but do not typically infiltrate through the BM into the dermis. Conversely, BM‐like material can be seen around invasive carcinoma cells and around metastatic tumour cell deposits. Here, we review the role of MECs and BM in breast pathology and highlight potential clinical implications. We advise caution in interpretation of MEC features in breast pathology and mindfulness of the substantive evidence base in the literature associated with behaviour and clinical outcome of lesions classified as benign on conventional morphological examination before changing classification to an invasive lesion on the sole basis of MEC characteristics.     

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign lesions and ductal carcinomas in the human breast

Tenascin (TN)-C and fibronectin (FN), which are glycoproteins of the extracellular matrix (ECM), are up-regulated in cancer tissues, including breast cancer. For assessment of their involvement in cancer invasion, it is important to know which cells are responsible for their production and secretion. The distribution of cells expressing TN and FN mRNAs in benign and malignant human breast tissues was therefore analysed by in situ hybridization, using digoxigenin-labelled cRNA probes, in addition to demonstrating the proteins immunohistochemically. Both mRNAs were expressed in epithelial cancer as well as in stromal cells in a large fraction of the tumours, with co-expression in individual cells. In cancers with intraductal components and/or those consisting of large nests, the mRNAs were more often expressed in the cancer than in the stromal cells. In scirrhous carcinomas, in contrast, the stromal cells were almost always positive for TN and FN mRNAs, while the cancer cells only rarely exhibited TN or FN expression. In benign lesions including adenosis, fibroadenoma and intraductal papilloma, the expression patterns also varied. These findings indicate that TN and FN co-expressed by cancer cells and stromal cells are probably involved in the intraductal extension and early invasion of cancer cells and in the remodelling of cancer stroma. © 1997 John Wiley & Sons, Ltd.     

Loss of types XV and XIX collagen precedes basement membrane invasion in ductal carcinoma of the female breast

Ductal and lobular carcinomas comprise most malignancies of the female breast and the morbidity and mortality associated with breast cancer. During the progression from in situ to invasive stages, tumour cells penetrate the epithelial and vascular basement membranes (BM) to realize full metastatic potential. While the definition of these structures has primarily resulted from analysis of laminin and type IV collagen, characterization of newly discovered BM/BM zone (BMZ) proteins will further elucidate the interactions between tumour cells and the host stroma. We have studied the expression of two non-fibrillar BMZ collagens, the type XV proteoglycan and collagen XIX, in breast cancer where a linear, well-formed BM becomes fragmented and even lost in the progression of epithelial malignancy. In the normal breast, types XV and XIX were found in all BMZ: epithelial, muscle, neural, endothelial, and fat. In in situ lesions, these two collagens, and particularly type XV, were often absent from the BM/BMZ displaying a continuous or just focally disrupted type IV/laminin staining pattern. In contrast, infiltrating ductal carcinomas showed only rare traces of laminin and collagen IV reactivity adjacent to the glands and tumour nests, and similarly there was little if any evidence of types XV and XIX collagen. All four molecules were, however, detected in the interstitium associated with some of the invasive carcinomas. The data suggest that types XV and XIX collagen are lost early in the development of invasive tumours, prior to penetration and eventual dissolution of the epithelial BM. Disappearance of these proteins from the BM/BMZ may signal remodelling of the extracellular matrix to promote tumour cell infiltration.     

Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers

Integrin dimer α₆β₄ is a transmembrane component of an epithelial cell adhesion complex that consists of hemidesmosomes (HDs), basement membrane (BM)-associated laminin-5 (Ln-5), and anchoring filaments/type VII collagen, all of which are absent from normal thyroid follicular epithelium. In the present study, the expression of epithelial cell adhesion complex antigens in thyroid tumours was investigated using immunohistochemistry. In addition to integrin subunits α₆ and β₄, immunoreactivity was found for all chains of Ln-5, α3, β3 and γ2, type VII collagen and hemidesmosomal antigen, HD1, in most thyroid carcinomas associated with tumour anaplasia and papillary growth pattern and located at the border of parenchymal cells and connective tissue or blood vessel walls. In addition, a more restricted expression of bullous pemphigoid antigens 180 and 230 (BP180 and BP230), constituents of HDs, was found in some papillary and anaplastic carcinomas and atypical adenomas. Adhesion complex antigens were located to regions of cells which were immunoreactive for cytokeratin (ck)-5 and proliferating cell nuclear antigen Ki-67. The results suggest that in thyroid carcinomas, the emergence of adhesion complex antigens is associated with squamous differentiation and high proliferative activity. © 1998 John Wiley & Sons, Ltd.     

Bone-derived soluble factors and laminin-511 cooperate to promote migration,invasion and survival of bone-metastatic breast tumor cells

Abstract

Tumor intrinsic and extrinsic factors are thought to contribute to bone metastasis but little is known about how they cooperate to promote breast cancer spread to bone. We used the bone-metastatic 4T1BM2 mammary carcinoma model to investigate the cooperative interactions between tumor LM-511 and bone-derived soluble factors in vitro. We show that bone conditioned medium cooperates with LM-511 to enhance 4T1BM2 cell migration and invasion and is sufficient alone to promote survival in the absence of serum. These responses were associated with increased secretion of MMP-9 and activation of ERK and AKT signaling pathways and were partially blocked by pharmacological inhibitors of MMP-9, AKT-1/2 or MEK. Importantly, pre-treatment of 4T1BM2 cells with an AKT-1/2 inhibitor significantly reduced experimental metastasis to bone in vivo. Promotion of survival and invasive responses by bone-derived soluble factors and tumor-derived LM-511 are likely to contribute to the metastatic spread of breast tumors to bone.     

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell linein vitro andin vivo

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experimentsin vivo andin vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluationin vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DR090-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membranein vitro.     

Oestrogen receptors in benign epithelial lesions and intraduct carcinomas of the breast: an immunohistological study

We examined 198 breast lesions, representing commonly encountered benign epithelial proliferative disorders, lobular carcinoma in situ and intraduct carcinoma, immunohistologically for oestrogen receptors (ER). A mixture of three ER monoclonal antibodies--H222, D75 and D547--was used on sections of routinely processed and paraffin-embedded tissue blocks. Over 65% of the benign and malignant lesions showed some evidence of ER expression and significant staining was recorded by two observers in 28-31% of fibroadenomas, 18-28% of ductal epithelial hyperplasias, 30-40% of sclerosing adenosis cases, 38-45% of papillomas, 60% of in situ lobular carcinomas and 42-45% of intraduct carcinomas. Apocrine metaplastic cells and myoepithelial cells showed absent or only weak staining. Amongst intraduct carcinomas, less than 20% of comedo carcinomas and over 50% of cribriform, papillary and solid variants showed significant ER staining.     

Expression of the 67 kD laminin receptor in human cervical preneoplastic and neoplastic squamous epithelial lesions: an immunohistochemical study

Interactions of cancer cells with laminin play a critical role during the progression of solid malignant tumours. Increased expression of the 67 kD laminin receptor (67LR), one of the several laminin binding proteins, is associated with the invasive and metastatic capacity of various types of cancer, including breast, colon, ovary, lung, and endometrial carcinoma. In this study, 67LR expression was analysed in a series of cervical biopsy specimens including 16 normal cervical tissues, 36 low-grade squamous intraepithelial lesions (SILs), 24 high-grade SILs, and 11 invasive carcinomas. Detection of the 67LR was performed using immunoperoxidase staining and the monoclonal antibody MLuC5 which specifically recognizes the 67LR. Immunostaining of the 67LR was correlated with human papillomavirus (HPV) type detected by in situ hybridization and with proliferative activity of the lesion determined by immunohistochemistry with the MIB-1 monoclonal antibody, specific for the Ki67 antigen. Increased expression of the 67LR was correlated with the histological severity of the lesions, with the strongest immunoreactivity being found in invasive carcinomas. Significant differences in 67LR expression were found between normal cervical epithelium and high-grade SILs (P<0·05, non-parametric Mann-Whitney test) or invasive carcinomas (P<0·001), as well as between low- or high-grade SILs and invasive carcinoma (P<0·01 and P<0·05, respectively). Ki67 antigen expression also increased with the severity of the lesions. There was a positive correlation for each type of lesion between expression of the 67LR and of the Ki67 antigen. No specific relationship was found between 67LR or Ki67 antigen immunostaining and HPV type detected in SILs, segregated into low-grade and high-grade lesions. These data add weight to the evidence that increased expression of the 67LR is a consistent, but not sufficient feature of the invasive and metastatic phenotype and suggest that high expression of the 67LR might be associated with both more proliferative and more aggressive cervical (pre)neoplastic lesions. © 1997 John Wiley & Sons, Ltd.     

Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistochemical study with antibodies to type IV collagen and laminin

Patterns of basement membrane deposition were investigated in benign and malignant naevo-melanocytic lesions using antibodies to type IV collagen and laminin. Paraffin sections required pretreatment with 6 M guanidine-HCl in addition to pepsin pretreatment. Basement membrane deposition was found around clusters as well as individual naevo-melanocytic cells in contact with dermal stroma. However, between keratinocytes and intra-epidermally located naevo-melanocytic cells, basement membrane immunostaining could not be detected. Tumour cell-stromal interaction is apparently a prerequisite for basement membrane deposition in naevo-melanocytic lesions. Basement membrane discontinuities, in the absence of inflammatory infiltrate, appeared, in doubtful cases, to be evidence in favour of malignant melanoma. The general pattern of basement membrane deposition in benign and malignant lesions was found to be similar and therefore of no help in differential diagnosis. Identification of hyaline bodies, which show immunoreactivity with antibodies to basement membrane components, may be helpful in distinguishing between Spitz naevi and malignant melanomas. Detection of vascular invasion, a prognostic indicator in malignant melanoma, is facilitated by basement membrane immunostaining.     

Angiogenesis as a prognostic marker in breast carcinoma with conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis

It has now been clearly established that quantitative immunohistochemical methods applied to tumour angiogenesis under suitable quality control conditions are a powerful prognostic tool for use in the initial assessment of breast carcinomas. Appropriate parameters for predicting the aggressiveness of tumours and their sensitivity to treatment are, however, still required. To determine whether the microvessel count (MVC) may serve to predict the chemotherapeutic response, a retrospective study was carried out on a series of 162 patients with breast carcinoma, who were all treated with the same standard adjuvant chemotherapy. Angiogenesis was assessed by performing CD31 immunostaining and MVC per mm². Several other factors such as P53, ERBB2, BCL2, and Ki67 were also measured, and their prognostic value was compared with that of the MVC. The MVC was not found to be correlated with any of the other prognostic parameters, but turned out to be of great prognostic value whatever the threshold value chosen, which suggests that it is continuously valid at all levels. The median value of the MVC (43·5 per mm²) divided this series into two significantly different prognostic categories, in terms of both disease-free survival (P=0·0002) and overall survival (P=0·037). Univariate analysis showed that most of the parameters analysed were of prognostic value regarding the disease-free survival, namely grade (P=0·029), mitotic index (P=0·049), size (P=0·015), oestrogen receptors (P=0·022), progesterone receptors (P=0·018), P53 (P=0·0045), ERBB2 (P=0·046), and Ki67 (P=0·0008). As regards overall survival, grade and ERBB2 showed a loss of prognostic value. In multivariate analysis on disease-free survival, the MVC was the most accurate prognostic factor (RR=2·64), followed by Ki67 (RR=2·06) and P53 (RR=1·69). With respect to overall survival, the MVC ranked third among the prognostic parameters analysed. Standard chemotherapy did not reduce the high prognostic value of the MVC performed on tumour angiogenesis. This suggests that the MVC may predict the degree of resistance to chemotherapy. Patients with high levels of angiogenesis, particularly node-negative patients, might therefore be able to benefit from adjuvant therapy of another kind. © 1998 John Wiley & Sons, Ltd.     

Mucinous lesions of the breast: potpourri of old and new

Mucinous lesions of the breast encompass a range of benign and malignant entities characterized by extracellular mucin production. Increased sampling of mammary calcifications has identified a range of mucocele-like lesions, which are associated with benign proliferative and atypical intraductal epithelial proliferation ranging in architectural complexity from flat epithelial atypia to ductal carcinoma in situ. Mucinous carcinoma is a unique histologic subtype of breast cancer with a good prognosis. Mucinous carcinoma, especially in males and older females, can arise in association with solid papillary carcinoma. Recent molecular studies reveal distinct genomic changes in mucinous carcinoma. Micropapillary mucinous carcinoma is a recently described histologic variant of breast cancer with aggressive clinical features and poorer outcome. Not all mucinous lesions are of ductal epithelial phenotype; rarely, lobular carcinoma can present with extracellular mucin. This concise review discusses clinical features, histological findings, and differential diagnoses of mucinous lesions of the breast, including common and some rare lesions as well as recently described entities.     

MicroRNA-130a inhibits cell proliferation,invasion and migration in human breast cancer by targeting the RAB5A

MiR-130a has been demonstrated to play important roles in many types of cancers. Nevertheless, its biological function in breast cancer remains largely unknown. In this study, we found that the expression level of miR-130a was down-regulated in breast cancer tissues and cells. Overexpression of miR-130a was able to inhibit cell proliferation, invasion and migration in MCF7 and MDA-MB-435 cells. With the bioinformatics analysis, we further identified that RAB5A was a directly target of miR-130a, and its mRNA and protein level was negatively regulated by miR-130a. Immunohistochemistry verified RAB5A was upregulated in breast cancer tissues. Therefore, the data reported here demonstrate that miR-130a is an important tumor suppressor in breast cancer, and imply that miR-130a/RAB5A axis have potential as therapeutic targets for breast cancer.     

Patterns of basement membrane deposition in benign and malignant breast tumours

We have examined epithelial basement membranes in tissue samples of seven normal breasts, 64 benign breast lesions and 63 malignant breast tumours by immunocytochemistry, using polyclonal antisera specific for type IV collagen. In normal breast tissue as well as in all benign tumours a continuous basement membrane was found at the epithelial stromal interface. In benign proliferative lesions, epitheliosis and papillomatosis could be more accurately distinguished with basement membrane staining. This approach also facilitated the differentiation between sclerosing adenosis and tubular carcinoma, since the tubules in sclerosing adenosis are surrounded by a continuous basement membrane whereas in tubular carcinoma basement membranes are almost entirely absent. In radial scar lesions the tubules were always surrounded by intact basement membranes, which underlines the fact that these are benign lesions. In breast carcinoma we could not detect a relationship between histological grade and the extent of basement membrane deposition. However, in different tumour types the basement membrane alterations varied. In infiltrating lobular carcinoma of the alveolar type, fragments of basement membrane were found, whereas in the classic and trabecular type, basement membranes were absent, suggesting that the alveolar type may be an intermediate phase in the progression of lobular carcinoma in situ to infiltrating lobular carcinoma. It is concluded that basement membrane immunocytochemistry, using antibodies to type IV collagen, is useful in the differentiation between benign and malignant breast lesions and in the classification of breast neoplasms.     

Primary breast myoepithelial cells exert an invasion-suppressor effect on breast cancer cells via paracrine down-regulation of MMP expression in fibroblasts and tumour cells

In normal breast and ductal carcinoma in situ, myoepithelial cells form an incomplete layer separating the epithelial compartment from the stromal environment. Transition to invasive disease is marked by penetration of the myoepithelial-basement membrane (BM) interface. One mechanism involved in tumour invasion is breakdown of extracellular matrices by matrix metalloproteinases (MMPs). It was hypothesized that myoepithelial cells may modulate tumour invasion by controlling MMP gene expression, both in tumour cells and in peri-ductal fibroblasts. To investigate this, myoepithelial cells from normal breast were purified and characterized and their effect on tumour cell invasive potential was assessed. The effect on MMP gene expression of breast cancer cells cultured alone or in combination with primary normal breast fibroblasts was also analysed using RT-PCR with ELISA quantitation, with zymographic analysis to measure enzyme activity. Normal breast myoepithelial cells significantly reduced invasion by the breast cancer cell lines MCF-7, T47D, MDA-MB 231, and MDA-MB 468 when they were cultured alone or in the presence of a fibroblast population. Reduced invasion was associated with changes in MMP gene expression. In those tumour cells expressing MMP, there was a significant down-regulation of MMP-2 (MDA-MB 468, p<0.001), MMP-9 (MDA-MB 231, p=0.05; MDA-MB 468, p<0.001), and MT1-MMP (p<0.001 for both MDA-MB 231 and MDA-MB 468). Myoepithelial cells also caused a significant decrease in MMP gene expression in co-cultured fibroblasts. Furthermore, this was associated with reduced gelatinolytic activity as identified by zymography. This study demonstrates for the first time that primary myoepithelial cells from normal breast reduce breast cancer cell invasion and that this is mediated via modulation of both tumour cell and fibroblast function. This emphasizes the importance of the myoepithelial cell in controlling the breast microenvironment and focuses on the potential significance of the loss of this population with disease progression.     

Expression of epithelial markers in sarcomatoid carcinoma: an immunohistochemical study

Thirty-four cases of sarcomatoid carcinoma with minimal epithelial components (SC) and six cases of sarcomatous tumour without any epithelial component (ST) in various organs were studied by the immunoperoxidase technique for the expression of epithelial markers, cytokeratins and epithelial membrane antigen (EMA). Employing antibodies against both high and low molecular weight cytokeratins, sarcomatoid components in 30 examples of SC were stained positively. Epithelial membrane antigen was demonstrated in 19 out of 34 SC. The positive cells for epithelial markers within sarcomatoid components in some cases of SC, which were regarded as originating from squamous cell carcinoma, tended to be seen less frequently than in the tumours derived from adenocarcinoma or transitional cell carcinoma. In six cases of ST, stain for EMA was negative and stain for cytokeratins was positive in three examples. The immunohistochemical examination of epithelial markers in the tumours of these types may be of value in differentiating these tumours from true sarcomas.     

Effect of MRC-5 fibroblast conditioned medium on breast cancer cell motility and invasion in vitro

We used Transwell chambers to study separately cellular motility and invasion. In order to assess the cellular motility, polycarbonate microporous filters were coated with extracellular matrix proteins which adsorbed on the filters without clogging the pores. To investigate the invasive behavior of tumor cells, filters were covered with a layer of Matrigel which clogged the pores. The motility and the invasion of breast cancer cell lines (MDA-MB-231, MCF-7/6 and MCF-7/AZ cells) were assessed quantitatively in different culture media: defined (serum-free), serum-containing and normal human fibroblast MRC-5 conditioned media. Inserum-containing medium, tumor cells migrated and invaded through the coated and covered filters. Their motility and invasion potentials were considerably lower in defined medium, whereas medium conditioned by MRC-5 fibroblasts stimulated both motility and invasion but not growth. The MRC-5 conditioned medium induced also the spreading of clusters of MCF-7 /6 cells grown on Matrigel-coated plates. © Rapid Science 1998     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.