http://www.jbc.org/content/254/19/9933.full.pdf

Laminin--a glycoprotein from basement membranes.

Tumour cells traverse epithelial and endothelial basement membranes during the successive stages of the metastatic process. At the transition from in situ to invasive carcinoma, local dissolution of the basement membrane is observed microscopically1,2, and coincides with tumour cell invasion of the underlying stroma. Tumour cells further traverse the endothelial basement membrane during entry into and egress from blood vessels3–5. Electron microscopic studies have shown local dissolution of basement membrane at its area of contact with invading tumour cells, suggesting an enzymatic mechanism3,6,7. Basement membranes are resilient structures which present a mechanical barrier to invasion8. Type IV collagen is a major structural protein of basement membranes and is chemically and genetically distinct from stroma collagen types I and III and cartilage collagen type II9,10. Previously characterised animal collagenases which cleave collagen types I; II and III fail to degrade type IV collagen11,12. We have recently purified about 1,000-fold and characterised a neutral protease activity preferential for type IV collagen from metastatic tumour cells and shown that it (1) produces specific degradation products, (2) has a molecular weight of 65,000, (3) is not plasmin or a cathepsin, by pH and inhibitor studies, and (4) does not significantly degrade other collagens or fibronectin12,13. Here we extend the relevance of this finding by quantitating the ability of several murine tumour cell lines of known metastatic potential to degrade type IV collagen. The cell lines with the highest incidence of spontaneous metastasis exhibit the greatest level of type IV collagen-degrading activity in two different assays using either living cells or media obtained from cell cultures.

Metastatic potential correlates with enzymatic degradation of basement membrane collagen.

In recent years, the basement membrane (BM)--a specialized form of extracellular matrix (ECM)--has been recognized as an important regulator of cell behaviour, rather than just a structural feature of tissues. The BM mediates tissue compartmentalization and sends signals to epithelial cells about the external microenvironment. The BM is also an important structural and functional component of blood vessels, constituting an extracellular microenvironment sensor for endothelial cells and pericytes. Vascular BM components have recently been found to be involved in the regulation of tumour angiogenesis, making them attractive candidate targets for potential cancer therapies.

Basement membranes: structure, assembly and role in tumour angiogenesis

We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.

Basement Membrane Complexes with Biological Activity

Matrigel: basement membrane matrix with biological activity.

We have studied a murine tumor previously classified as a poorly differentiated chondrosarcoma. Although the cells in this tumor are surrounded by large quantities of extracellular matrix material, the matrix fails to react with stains specific for the sulfated glycosaminoglycans present in normal cartilage. Here we show at the ultrastructural level that the tumor matrix is a homogeneous, nonfibrillar material, resembling basement membrane. Neither the proteoglycan matrix granules nor collagen fibrils characteristic of cartilage are present in the tumor matrix. Amino acid analyses of whole tumor tissue, enzyme-solubilized tumor components, and the protein extracted from lathyritic tumors confirmed that the tumor matrix is a basement membrane collagen. The collagenous protein extracted from the tumor by nonenzymatic means contains three unique polypeptides larger than the alpha-chain components of the other types of collagen. These studies indicate that the tumor is not a type of chondrosarcoma but a basement membrane producing tumor.

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E B McGoodwin

Papers

A murine tumor producing a matrix of basement membrane