Basement membranes in adenoid cystic carcinoma. An immunohistochemical study.

Tissue samples from 30 patients with adenoid cystic carcinoma and 20 with adenocarcinoma of salivary gland origin were studied by immunohistochemical staining with specific antibodies to the four macromolecules that are present in normal basement membranes: type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. In the adenoid cystic carcinoma samples, the four proteins were localized in different types of extracellular matrices in the tumor, namely pseudocystic spaces, hyaline stroma, and around tumor cell nests. The staining intensity was enhanced by pretreatment with hyaluronidase. The tumor cells of adenoid cystic carcinoma showed a tendency to proliferate with individual cells in contact with the basement membrane and to infiltrate through basement membrane-rich tissues, such as peripheral nerves, blood vessels, and skeletal muscles. In contrast, only circumferential staining of tumor cell nests was obtained in adenocarcinoma samples. The results suggest that adenoid cystic carcinoma is a tumor with affinity for basement membranes, and this basic feature is reflected in its histology and presumably in its biologic behavior. Immunostaining with antibodies to basement membrane proteins appears to be useful for differential diagnosis of some types of these two carcinomas.     

Dynamic alterations of the extracellular environment of ovarian surface epithelial cells in premalignant transformation,tumorigenicity, and metastasis

BACKGROUND: Ovarian surface epithelial cells are positionally organized as a single cell layer by a sheet of basement membrane. It is believed that the contact of the ovarian surface epithelial cells with the basement membrane regulates cell growth and ensures the organization of the epithelium. Disabled-2 (Dab2), a signal transduction protein and a candidate tumor suppressor of ovarian carcinoma, functions in positional organization of ovarian surface epithelial cells. In ovarian carcinomas, genetic and epigenetic changes enable the tumor cells to escape positional control and proliferate in a disorganized fashion. Alterations in the extracellular environment may also be critical for tumor initiation and progression. METHODS: We analyzed and compared the presence of collagen IV and laminin, the scaffold proteins of the basement membrane, and Dab2 in 50 ovarian tumors that are restricted to the ovaries and in 50 metastases of ovarian tumors by immunohistochemistry. Expression of collagen IV, laminin, and Dab2 was also analyzed by Northern blotting in a panel of human ovarian surface epithelial and cancer cell lines. RESULTS: The basement membrane is often absent in morphologically benign ovarian surface and cyst epithelium and low-grade tumors and collagen IV and laminin are absent in the extracellular matrix of most of the primary tumors tested. Of the 50 ovarian tumors confined to the ovaries, 6% (3 of 50) were collagen IV positive and 24% (12 of 50) were laminin positive tumors. Of the 50 metastatic tumors, 16% (8 of 50) are collagen IV positive and 86% (43 of 50) are laminin positive. In addition, even in the metastatic ovarian tumors that are largely collagen IV negative, there are pockets of local areas in which the tumor cells are surrounded by collagen IV-positive staining. Dab2 is absent in the majority of ovarian tumors found in both ovaries and metastatic sites. In both nontumorigenic human ovarian surface epithelial and cancer cell lines, collagen IV, laminin, and Dab2 are expressed aberrantly. CONCLUSIONS: Loss of the basement membrane may be an early event in the preneoplastic transformation of ovarian surface epithelium and in the early stages of tumorigenesis before tumor invasion and metastasis. The majority of primary ovarian tumors examined lack collagen IV and laminin in their extracellular matrix. However, expression of laminin is restored in the majority of metastatic tumors. Reexpression of collagen IV may also contribute to tumor metastasis. The ability of tumor cells to dynamically alter the expression of collagen IV and laminin may facilitate the shedding of cancer cells into the peritoneal spaces and subsequent attachment to the metastatic sites. We propose that loss of collagen IV and laminin may be an initial event in ovarian tumorigenicity and that restoration of collagen IV and laminin expression in the later stages of tumor development may promote metastasis of ovarian tumors.     

Degradation of basement membrane by murine tumor cells.

Tumor cells from the murine T241 fibrosarcoma, which rapidly and reproducibility produces pulmonary metastases, were tested in vitro for their ability to degrade isolated pulmonary basement membrane. Degradation of basement membrane substrate was quantified by the culture of the substrate with tumor cells and measurement of the solubilized hydroxyproline and hexose glycoprotein at neutral pH. It was found that tumor cells collected in the tumor venous drainage were associated with a significantly greater solubilization of basement membrane than were tumor cells obtained from the primary tumor mass. Tumor cells were also assayed for their ability to solubilize type I collagen purified from human dura. Venous effluent tumor cells solubilized collagen to a significantly greater level than primary tumor cells, spleen cells, or liver cells. These findings raised the possibility that metastasizing tumor cells may be a distinct tumor subpopulation with regard to invasive potential.     

Extracellular matrix proteins characterize human tumor cell lines

Extracellular matrix proteins synthesized and secreted by adherent human tumor cell lines were analyzed using metabolic labelling with glycine and proline in the presence of ascorbate, polypeptide analysis and polyacrylamide gel electrophoresis, affinity chromatography, collagenase digestion, and immunofluorescence staining. The results showed a characteristic pattern of matrix proteins for each tumor cell type. Tumor cell lines of mesenchymal origin produced mostly interstitial types (I and II) of collagen and fibronectin. Carcinoma cell lines secreted only basement membrane proteins, type IV collagen, laminin and fibronectin, but not interstitial collagen. A melanoma and a rhabdomyosarcoma cell line produced type V of procollagen that has not previously been described in cell culture. Neuroblastoma cells were shown to be phenotypically heterogeneous also with respect to matrix protein production. We propose that the analysis of extracellular matrix proteins may serve as an adjunct in the classification of human tumors.     

A specific antigenic defect of the basement membrane is found in basal cell carcinoma but not in other epidermal tumors

The basement membrane of basal cell carcinoma was characterized by indirect immunofluorescence using antibodies to laminin, type IV collagen, and bullous pemphigoid antigen, three distinct protein components of basement membrane. Aggregates of basal cell carcinoma in the dermis were surrounded by a continuous basement membrane containing laminin and type IV collagen; however, bullous pemphigoid antigen was either completely undetectable or faint and discontinuous rather than linear. In contrast to this specific defect in bullous pemphigoid antigen found in basal cell carcinoma, well-differentiated superficially invasive epidermal squamous cell carcinoma and several benign epidermal tumors (trichoepithelioma, wart, keratocanthoma, seborrheic keratosis, cylindroma) displayed all three antigens in the basement membrane that surrounded epithelial cell aggregates. Cylindroma displayed in addition, broad areas of type IV collagen in the hyalinized material between epithelial islands. Basal cell carcinoma was the only tumor examined in which there was a specific antigenic defect in the basement membrane. This defect in bullous pemphigoid antigen may be due to abnormal synthesis by the tumor cells and could be related to the absence of differentiation of these cells.     

Dentinogenic ghost cell tumor: histologic aspects, immunohistochemistry, lectin binding profiles, and biophysical studies

Dentinogenic ghost cell tumor accompanied with calcifying odontogenic cyst (COC) was described in terms of its clinical, histological, immunohistochemical, lectin binding and biophysical properties. The case was a 38-year-old Japanese female, in whom the tumor had arisen in the right mandibular premolar and molar region. Material obtained by partial mandibulectomy was used. Decalcified paraffin sections were used to detect keratins, involucrin, and lectin binding; and non-decalcified thin sections were used for biophysical analysis. The lesion comprising dentinogenic ghost cell tumor and COC contained odontogenic epithelium with ghost cells, eosinophilic amorphous materials and osteodentin. Some of the eosinophilic material had undergone transformation into osteodentin. Keratins in odontogenic epithelia showed positive PKK1 staining in peripheral tumor cells, and stainings with KL1 and involucrin were positive in centrally located cells. Lectin binding in the amorphous materials was comparatively strong for PNA, and SBA, moderate for WGA, RCA-1, and UEA-1, and slight for DBA and ConA. Lectin binding affinities were higher in the amorphous materials than in the osteodentin. Elemental analysis with an electron probe X-ray microanalysis of the amorphous materials and osteodentin showed a pattern similar to that found in the normal dentin. The biologic properties of the eosinophilic amorphous materials suggested the material to be poorly calcified osteodentin, which gradually transformed into the well-calcified type.     

Significant correlation between the presence of type IV collagen in the duct wall and the development of wide intraductal cancerous extension in breast cancer

To identify the characteristics of cases with an wide intraductal cancerous extension (WICE), we examined the relationship between WICE and type IV collagen distribution, and the relationship between WICE and the content of the proliferation-associated proteins in human breast cancer. The immunohistochemical distribution of type IV collagen and proliferating cell nuclear antigen (PCNA) were investigated in formalin-fixed tissue sections from 21 breast cancer cases. We demonstrated a significant correlation (p=0.014) between the presence of WICE and immunostaining of type IV collagen in the cancerous ducts (ducts occupied by cancer cells) in the central invasive area of breast cancer. However, no correlation was found between the presence of WICE and the PCNA index (percentage of positive cells per 1000 tumor cells). These findings suggest that the lack of the process of the loss of type IV collagen in the duct wall is more important than the nature of the tumor cells in the development of WICE.     

Distribution of myoepithelial cells and basement membrane proteins in the normal breast and in benign and malignant breast diseases

An immunocytochemical method for fixed and paraffin-embedded human breast biopsies is reported for the detection of myoepithelial and epithelial cells using antibodies to myosin and keratin, respectively, and of basement membranes using antibodies to laminin and type IV collagen. Using these markers, myoepithelial cells can be clearly distinguished in the normal breast and in the benign breast diseases sclerosing adenosis, epitheliosis, and fibroadenoma. In sclerosing adenosis, myoepithelial cells form a major cellular component. A stromally derived spindle cell is identified which stains with myosin but not with keratin antibodies (myofibroblast). These cells are seen in one-fifth of the fibroadenomas. Although cells staining with myosin antibodies are seen in the infiltrating component of all 18 carcinomas examined, elongated cells staining with both myosin and keratin antibodies (myoepithelial-like) are seen in only one infiltrating carcinoma where they are interposed at the stromal-epithelial junction of the infiltrating tumor cells. In contrast to the situation in benign breast diseases, mature myoepithelial cells form a very minor component of the majority of infiltrating ductal carcinomas. Basement membrane proteins, laminin, and type IV collagen are present in normal breast, benign breast disease, and grade I infiltrating ductal carcinomas but are absent in carcinomas of grades II and III.     

Type IV collagenases in invasive tumors

Summary The matrix metalloproteinases appear to be elevated in tumors with metastatic potential, and may well be involved in penetration of the basement membrane and degradation of extracellular proteins including type IV collagen. An imbalance between the 72 kDa and 92 kDa type IV collagenases and the associated tissue inhibitors of these metalloproteinases (TIMPs) may therefore have a role in the invasive phenotype. Cultured tumor cells with invasive potential secrete both type IV collagenases, though in tumors there is some evidence that the 72 kDa form at least may be produced by stromal cells at the invading tumor front rather than primarily by the tumor cells themselves, while the 92 kDa form may be synthesized in macrophages near the front. These collagenases are elevated in invasive as compared within situ tumor components, but their specific roles and prognostic significance are not yet established.     

Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.     

Clearing and release of basement membrane proteins from substrates by metastatic tumor cell variants

A variety of normal cells and tumor cell lines of differing metastatic potential were evaluated for their effect on various substrates consisting of purified preparations of the basement membrane-associated proteins fibronectin, laminin, and type IV collagen, which were labeled with tritium and/or rhodamine isothiocyanate. Different degrees of clearing or release of material from the substrate were observed, depending on the cell-protein combination. Normal fibroblasts as well as transformed cells and cells of low metastatic potential showed extensive clearing of surfaces coated with fibronectin, laminin, and type IV collagen. This clearing of protein began at sites of initial cell adhesion and was restricted to areas beneath and/or along the apparent paths of cell migration and beneath cellular processes. Covalent attachment of adhesion proteins to glass coverslips nearly eliminated clearing and release. Studies showed that a substantial amount of the fibronectin released from the substrate by HT-1080 fibrosarcoma cells is due to proteolysis. Release and clearing of substratum-attached protein is reduced by approximately 50% by antibodies specific for the protein on the substrate. Tumor cells with low metastatic potential were found to produce higher levels of clearing and release of protein adsorbed to the substrate than tumor cells with high metastatic potential. This was true for variants of the murine K-1735 melanoma and the UV-2237 fibrosarcoma with high and low metastatic capability on all three basement membrane-associated protein substrates. The differences in clearing and release between high and low metastatic cells were not due to differences in initial cell adhesion to the substrates but may be associated with differences in the affinity, type of cell-substrate interactions, proteases, or other variables.     

Stages of neoplastic transformation of human breast tissue as monitored by dissolution of basement membrane components. An immunoperoxidase study

Two constituents of basement membrane, type IV collagen and laminin, were studied by immunoperoxidase methods in a group of breast lesions, exhibiting a range of neoplastic transformation. In normal breast, fibroadenoma, sclerosing adenosis, intraductal hyperplasia, and intraductal carcinoma there was intact basement membrane surrounding the ducts and lobules, as evidenced by an extracellular linear staining pattern with antibodies to type IV collagen and laminin. In intraductal carcinoma with microinvasion, there was fragmentation and absence of the basement membrane at the areas of microinvasion. Infiltrating carcinoma and metastatic breast carcinoma were usually devoid of surrounding extracellular basement membrane containing type IV collagen and laminin. However, a few well-differentiated carcinomas showed scattered extracellular deposits of this matrix material. Individual metastatic carcinoma cells, such as those in lymph nodes, contained intense cytoplasmic immunoreactivity with these antibodies. These results support the concept of basement membrane degradation associated with invasion. Furthermore, at least some metastatic tumor cells retain the ability to synthesize laminin and type IV collagen, but do not exhibit an extracellular basement membrane. This may mean that the metastatic cells are degrading and/or failing to deposit the extracellular matrix.     

Adamantinoma of the tibia. An ultrastructural and immunohistochemical study

The light microscopic, ultrastructural, and immunohistochemical characteristics of two tibial adamantinomas are presented. The immunohistochemical studies utilized specific antibodies against Factor VIII-related antigen and keratin protein, considered as markers for endothelial and epithelial cells, respectively. These revealed positive staining for keratin in the tumor cells of both cases, whereas Factor VIII was not found in either. Ultrastructurally, both tumors had tonofilaments, desmosomes, gap junctions, microvillous-like projections, and basement membranes. Patient 1 had disease that was histologically of the classic spindle cell type; the disease of Patient 2 was atypical and closely resembled an epithelioid angiosarcoma. Immunohistochemical and ultrastructural findings in each case indicate an epithelial component in tibial adamantinoma.     

大肠癌分化,浸润和转移与细胞外基质的关系

应用SP免疫组化方法观察63例大肠癌细胞外基质成分层粘连蛋白（LN）、IV型胶原的表达。结果：部分癌组织显示胞浆LN阳性；LN与IV型胶原在基底膜的表达与分布基本一致，表现为基底膜不同程度的缺失，缺失程度与癌分化程度相关，分化越低，缺失越重；IV型胶原的缺失程度尚与癌的侵袭能力有关，浸润较深及有淋巴结转移者缺失较重。提示：LN与IV型胶原可成为临床判断大肠癌分化程度的有用指标；IV型胶原含量少的大肠癌，恶性度高，侵袭力强。     

Collagen biosynthesis in human neuroblastoma cell lines: evidence for expression of glial cell properties

An analysis was done on the synthesis of collagen, an extracellular matrix protein synthesized by Schwann cells, by several human neuroblastoma lines. Cultured cells were incubated in the presence of L-[2,3-3H]proline, and the collagens synthesized and secreted into the culture medium were analyzed by electrophoresis on acrylamide gels, ion exchange chromatography, and immunoprecipitation. The amount of collagen secreted by 4 cell lines tested represented less than 3% of the total protein synthesis, indicating a low degree of collagen biosynthesis by this tumor type. Analysis of collagen types secreted by all cell lines revealed the presence of a high-molecular-weight collagen precursor (Mr = 165,000) identified as type IV procollagen. In addition, several cell lines synthesized stromal type I and type III collagens. These studies show that neuroblastoma cells produce collagenous proteins including basement membrane collagen (type IV) and stromal collagens (types I and III), indicating that these cells express properties of glial cells such as Schwann cells.     

Directed migration of murine and human tumor cells to collagenases and other proteases

Tumor cell motility and the passage of tumor cells through various tissue matrices, including basement membrane, are important components of the metastatic process. Proteolytic enzymes, including a type IV collagen-specific collagenase, have been demonstrated to play a significant role in extracellular matrix and basement membrane degradation. In addition, exogenous collagenase has been shown to enhance the motility of some tumor cells independent of its effect on collagen-containing material. Previous studies have also indicated that collagen fragments are chemotactic for many tumor cells. We therefore studied the effect of type I and type IV collagen-specific collagenases, other enzymes involved in collagenase activation and connective tissue degradation, and subsequent collagen degradation products on the directed migration of tumor cells. We report that type I and type IV collagen-specific mammalian collagenases were potent chemoattractants as were native type I and type IV collagens and collagen fragments. Collagenase inhibitor SC44483 inhibited the type IV collagenase-stimulated migration. Collagenase pretreatment of the tumor cells potentiated the migratory response of the tumor cells to collagen and collagen fragments. The plasminogen activator, urokinase, as well as plasminogen itself also enhanced the directed migration of tumor cells in concentrations that suggest involvement of the appropriate cell surface receptor. The chemotactic response of tumor cells to the proteases studied extends the prior report of a role for collagenases and other matrix-active enzymes in tumor cell behavior in addition to matrix degradation.     

Distribution of laminin and collagen type IV in rat colon tumors induced by 1,2-dimethylhydrazine

Immunofluorescent distribution of basement membrane components laminin and collagen type IV was studied in 51 rat colon tumors induced by 1, 2-dimethylhydrazine. In normal colonic mucosa, adenomas and carcinomas in situ continuous basement membranes were present, while in adenocarcinomas they were altered to different extents. An uncoordinated loss, or dissociation, of the two markers studied was found: the degree of collagen type IV loss was often much higher than that of laminin in the same tumor. These data suggest that a reliable determination of cancer invasion by monitoring basement membrane alteration requires the use of several basement membrane markers.     

Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium

We have developed novel 3-dimensional in vitro and in vivo tissue models that mimic premalignant disease of human stratified epithelium in order to analyze the stromal contribution of extracellular matrix and basement membrane proteins to the progression of intraepithelial neoplasia. Three-dimensional, organotypic cultures were grown either on a de-epidermalized human dermis with pre-existing basement membrane components on its surface (AlloDerm), on a Type I collagen gel that lacked basement membrane proteins or on polycarbonate membranes coated with purified extracellular matrix proteins. When tumor cells (HaCaT-II4) were mixed with normal keratinocytes (4:1/normals:HaCaT-II4), tumor cells selectively attached, persisted and proliferated at the dermal-epidermal interface in vitro and generated dysplastic tissues when transplanted to nude mice only when grown in the presence of the AlloDerm substrate. This stromal interface was permissive for tumor cell attachment due to the rapid assembly of structured basement membrane. When tumor cells were mixed with normal keratinocytes and grown on polycarbonate membranes coated with individual extracellular matrix or basement membrane components, selective attachment and significant intraepithelial expansion occurred only on laminin 1 and Type IV collagen-coated membranes. This preferential adhesion of tumor cells restricted the synthesis of laminin 5 to basal cells where it was deposited in a polarized distribution. Western blot analysis revealed that tumor cell attachment was not due to differences in the synthesis or processing of laminin 5. Thus, intraepithelial progression towards premalignant disease is dependent on the selective adhesion of cells with malignant potential to basement membrane proteins that provide a permissive template for their persistence and expansion.     

Distribution of type IV collagen in benign and malignant epithelial proliferations. An indirect immunofluorescence study on the breasts, the lungs, and the skin

The distribution of type IV collagen in benign and malignant epithelial proliferations of the breasts, the lungs, and the skin was studied by an indirect immunofluorescence technique using specific antiserum. In benign lesions of the breasts, the staining for type IV collagen was present in all vascular and glandular basement membranes. In basal cell carcinoma of the skin, the basement membrane labeling was also found to be continuous. In malignant lesions of the breasts, the lungs, and the skin, staining for type IV collagen was seen only around well-differentiated glandular structures and in close contact to basal epidermal cells. This staining appeared as an irregular network. Of particular interest was the localization of type IV collagen in non-infiltrating lesions of the breasts and the bronchi where discontinuity in the basement membrane staining was observed. In contrast, there were no disruptions of basement membrane labeling in skin senile keratosis and in Bowen's disease. We conclude that the loss of type IV collagen seen in malignant proliferations in our study is related to overt or potential tumor cell infiltration and aggressiveness.