Glioblastoma Cell Adhesion and Invasion in Fibrin is Promoted through β₃ Integrin

Background: Glioblastoma is a highly aggressive brain tumor characterized by diffuse growth and resistance to therapy. Angiogenesis in glioblastomas is poorly organized and, therefore, tends to be associated with tumor cell necrosis, hemorrhage and thrombosis. This, in turn, leads to the formation of a fibrin-rich matrix, which could provide important adhesive cues for glioblastoma growth and proliferation.

Methods: Glioblastoma cell lines and primary tumor cells from patients with glioblastoma were embedded in a 3-dimensional matrix of clotted plasma, fibrin or Matrigel™ and scored for invadopodia formation as well as proliferation using phase contrast microscopy. Tumor stem cells were generated by culturing glioblastoma cells in the presence of Neurocult™ media. Tumor cells were plated on matrix-coated plates to assess adhesion and spreading.

Results: To better understand the diffusely infiltrating growth pattern of glioblastoma, we analyzed glioma cell adhesion in a panel of three glioblastoma cell lines. Featuring a spread and scattered phenotype, U87MG and U373MG glioblastoma cells efficiently attached to fibrinogen, fibronectin and collagen/laminin while the U343MG glioblastoma cell line, which resembled the cobble stone pattern of epithelial cell clusters, barely adhered to either of the ligands. The adhesive phenotype of the U87MG and U373MG cells correlated positively with integrin expression, lamellipodia formation and the capacity to form colonies in a clonogenic stem cell assay. Embedded in a 3-dimensional matrix of fibrin or clotted plasma U87MG and U373MG cells formed extensive invadopodia and grew at a rate that significantly outpaced the non-invasive U343MG cells. Notably, sprouting as well as nestin expression was more extensive in fibrin than in matrigel^TM when U87MG and U373MG cells were cultured in neural stem cell media suggesting that fibrin can bestow stem cell properties on glioblastoma cells. Blocking adhesive interactions between glioblastoma cells and the extracellular matrix with integrin β3 siRNA completely abolished invadopodia formation and caused a sustained growth inhibition in fibrin as well as matrigel that was phenocopied by knocking down fibronectin. These findings appear to be clinically relevant since freshly isolated tumor cells from patients with glioblastoma express more integrin β3 than primary tumor cells from patients with astrocytoma grade 2 or grade 3 and, at the same time, were significantly more proliferative in fibrin.

Conclusion: Together, these data demonstrate an important role of integrin β3 for infiltration and growth of glioblastoma cells in a 3D environment. Moreover, they suggest a specific role of blood clotting for promoting tumorigenic functions in high grade glioma.

No conflict of interest has been declared by the author(s).