The Influence of Human Umbilical Vein Endothelial Cells in the Formation of Glioblastoma Spheroids in Three-Dimensional Microwells



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Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with a high infiltrative capacity and increased vascularity. Despite current therapies overall patient survival rate is still poor. Therefore, alternative therapies have been considered for GBM treatment such as oncolytic viruses (OVs). The studies with two-dimensional (2D) cell cultures have been used as physiological models for understanding the cellular and molecular behavior of the GBM tumors. They are also highly desirable for virus studies. However they may not provide threedimensional (3D) characteristics of the tumors. Therefore, there is an increasing need in 3D cell culture models to investigate cell growth and cell-cell interactions. In this thesis, we hypothesized that culturing GBM cells with endothelial cells in the hydrogel microwells would mimic the in vivo microenvironment of tumor cells. Thus, we could provide beneficial 3D in vitro co-culture models for GBM treatment with oncolytic adenovirus, Delta-24-RGD (Δ24RGD). Therefore, in the first part of this study, we used our recently designed 3D PEGDA hydrogel microwell platform to co-culture GBM and HUVEC. We investigated the gene expression differences to gain further insight into molecular mechanism of 3D in vitro co-culture. Our gene expression data suggested the relative up-regulation of tumor angiogenesis markers PECAM-1/CD31 and VEGFR2, in co-cultures. Finally, we treated GBM tumors using Δ24RGD. Our results showed a significant cell lysis in 3D spheroids. The cell viability was decreased approximately 37%, 54% and 65% with 10, 50 and 100 MOIs, respectively. The infection of the Δ24RGD was found more effective on 3D spheroids when compared to 2D monolayer cell culture. These results implicate that our hydrogel microwell platform could contribute to mimic in vivo microenvironment of the GBM tumors and provide a promising 3D model to investigate the oncolytic potential of the viruses in vitro.



Glioblastoma, 3D spheroids