| Summary: | Little is known about the mechanisms which cause tumor cells to become invasive. For this thesis an in vitro tumor cell invasion assay was developed and used to investigate the role of a family of cell surface receptors, called integrins, in the invasion of tumor cells across basement membranes. It was also used to isolate an invasive cell line in order to study some of its properties.
Two osteosarcoma cell lines, HOS and MNNG-HOS, with known in vivo metastatic potentials were assayed in the in vitro invasion assay. Invariably, the highly tumorigenic and metastatic MNNG-HOS cells demonstrated greater invasive ability than the non-tumorigenic HOS cells. The chemical transformation of HOS into tumorigenic MNNG-HOS cells resulted in an increase in the expression of α₁β₁, α₂β₁ and α₆β₁ integrins which are laminin and collagen receptors. The expression of α₃β₁ and α₅β₁, were unchanged on MNNG-HOS cells and the expression of αvβ₃ was strongly downregulated on the more invasive cells. The invasion of HOS and MNNG-HOS cells through matrigel could be significantly inhibited when anti-fibronectin receptor or anti-α₆ subunit antibodies were present in the invasion assay, demonstrating the important role of integrins in tumor cell invasion.
The in vitro invasion assay described in this thesis was used to isolate a more invasive cell line from the prostate carcinoma cell line, PC-3, and called IPC-3. The morphology of these cells was distinct from the parent population, showing a spherical morphology as opposed to the triangular or spindle shaped morphology of PC-3 cells. These cells were also several times more invasive than the PC-3 cells and proliferated at a faster rate than the parent PC-3 cells. IPC-3 cells gradually lost their invasive potential after several months in tissue culture but retained their morphology and the characteristic expression of integrins. Adhesion of PC-3 and IPC-3 cells to purified extracellular matrix components revealed that IPC-3 attached well to laminin and to vitronectin. In adhesion kinetic experiments to purified extracellular matrix proteins, IPC-3 cells attached more quickly than PC-3 cells to larninin and vitronectin. Although the IPC-3 cells attached to the extracellular matrix proteins, fibronectin, vitronectin, laminin and collagen type IV, they were only able to spread on laminin and required several hours to do so. PC-3 cells also attached well to the extracellular matrix proteins but required only several minutes to spread on the matrix proteins including laminin. When plated on stock matrigel PC-3 cells organized themselves in tube-like structures while IPC-3 cells aggregated in clusters.
Analysis of the integrins on PC-3 and IPC-3 cells demonstrated that IPC-3 cells downregulated the expression of the α₁β₁,α₂β₁ and an almost completely downregulated the α₃β₁ integrin while the expression of the fibronectin receptor, α₅β₁,, and the vitronectin receptor, αⅴβ₁, were unchanged. The expression of α₆β₁, in both PC-3 and IPC-3 cells was not prominent. However the α₆β₄ receptor was present in large amounts and was upregulated in IPC-3 cells, particularly the 200 kDa subunit of β₄.
Immunofluorescence staining of PC-3 and IPC-3 cells demonstrated that PC-3 cells distributed their α₃β₁ and α₆β₄ integrin receptors mainly along the cell periphery and their αⅴβ₃ receptor in focal adhesion plaques, while the invasive IPC-3 cells concentrated their integrin receptors in circular adhesion structures.
Although much remains to be learned about integrins, they have an instrumental role in the invasion of tumor cells across basement membranes during the metastatic cascade of malignant cells. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate
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