(629c) Investigating the Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids

Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic three-dimensional (3D) spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells. We constructed 3D multicellular tumor spheroids (MCTS) using serum grown U87 GBM cells with C8-D1A mouse astrocytes or human umbilical vein ECs in low-attachment U-well plates. We characterized gene expressions of known stemness markers NES, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using quantitative real-time polymerase chain reaction (qRT-PCR). Specifically, we constructed coculture MCTS models by incorporating GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions and changing spheroid size dynamics over 7 days. We also observed an increased expression of stemness markers for GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively as compared to mixed monoculture spheroids (control). Further, we developed a triculture MCTS with GBM cells, astrocytes and ECs incorporated at a 1:4:9 ratio and found an increased expression of all the stemness markers. Overall, we demonstrated that astrocytes and ECs influenced the stemness marker expression of GBM cells in 3D MCTS, especially in higher coculture conditions with a larger number of astrocytes or ECs compared to tumor cells. This MCTS model, which enriches for the stemness marker expression, may provide an important tool for testing therapeutic strategies. Further exploration of the crosstalk between different cell types utilizing such models, would enhance our understanding of the GBM tumor microenvironment, eventually leading to the development of new therapeutic strategies.