Two-Step spreading mode of human glioma cells on fibrin monomer: Interaction of α(v)β₃ with the substratum followed by interaction of α₅β₁ with endogenous cellular fibronectin secreted in the extracellular matrix

Glioma cells, a human astrocyte-derived glioma cell line, were found to spread on immobilized fibrin monomer but not on fibrinogen. As a synthetic RGD-containing people GRGDSP blocked the spreading of glioma cells on fibrin monomer concentration-dependently, the spreading was thought to be mediated by their cell surface receptors. In fact, both the β₁- and β₃-integrins were located at 3 hours of incubation in the cytoplasmic areas and at 24 hours in the peripheral areas as well, although their distribution profiles were not necessarily identical with each other by immunohistochemical studies. By cytometry analysis utilizing respective monoclonal antibodies against α₅- and α(v)-integrins, we were able to show expression of α₅ (α₅β₁) but not α(v) on the surface of glioma cells at 24 hours of incubation on immobilized fibrin monomer. A 50-kDa transmembrane protein designated as integrin-associated protein (IAP) known to be closely associated with the β₃-integrin was also located in the cytoplasmic and apical areas of spreading glioma cells, but its specific antibody B6H12 failed to inhibit the spreading. Thus, the IAP-dependent involvement of β₃- integrin may not be predominantly involved in the glioma cell spreading on fibrin monomer. As an anti-α(v)β₃ antibody LM 609 inhibited the spreading of glioma cells partially at approximately 35%, the spreading seems to proceed in a two-step mode, i.e., via α(v)β₃ with its ligand exposed in fibrin monomer, and then via α₅β₁ with endogenous cellular fibronectin secreted from the glioma cells themselves. In fact, the cellular fibronectin was clearly visualized by confocal microscopic observation. Thus, upon contact with fibrin in clots formed at traumatized areas in the brain, for example, glioma cells may have a chance to adhere to and spread via α(v)β₃ with fibrin monomer and then via α₅β₁ with endogenous cellular fibronectin in the extracellular matrices.