Engineering a Novel 3D Tumor Microenvironment for Drug Testing



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Glioblastoma multiforme (GBM) is the most common malignant type of astrocytic tumor. GBM patients have a poor prognosis with a median survival of approximately 15 months due to high tumor recurrence and tumor resistance to chemotherapy despite the “Stupp” Regimen. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells mono-cultured in three-dimensional (3D) poly(ethylene glycol) dimethyl acrylate (PEGDA) microwells were co-treated with BAY 11-7082 and/or TMZ. Combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot, and immunofluorescence staining, were used to evaluate the effects of drugs on GBM cells. Moreover, our data showed that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating the actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment’s outcomes. Glioblastoma Multiforme (GBM) spheroids are embedded in a heterogeneous tumor microenvironment (TME) including astrocytes, endothelial cells, microglia, and pericytes, which play a role in GBM progression and resistance. To mimic the TME, we co-cultured GBM cells with human astrocytes (HA) and triple-cultured both commercial and patient-derived GBM cells with HA and human brain microvascular endothelial cells (HBMECs) in our 3D PEGDA microwells. In order to investigate the effect of the TME on GBM resistance to chemotherapeutic agents, we treated 3D spheroids with Bay 11-7082 and/or TMZ. Co-treatment applied to triple-cultured cells significantly changed the expression of some important genes such as glial fibrillary acidic protein (GFAP), vimentin, vascular endothelial growth factor receptor 2 (VEGFR2), platelet endothelial adhesion molecule1 (PECAM1/CD31), as well as Notch-1 and survivin, playing roles in GBM malignancy. To further investigate the role of HA and HBMECs on drug resistance to GBM, we examined the impact of HA- or HBMECs-Conditioned Media (CM) on GBM culture viability and gene expression. Our results showed that the presence of HA and HBMECs increased cell viability and the expression of the glial fibrillary acidic protein (GFAP), vimentin, Notch-1, and survivin in triple-cultures after the co-treatment. Furthermore, co-treatment induced apoptosis and reduced MGMT expression in GBM cells.



GBM tumor microenvironment, drug testing, hydrogel, PEGDA, NF-kB, TMZ, Astrocytes, the human brain microvascular endothelial cells.


Portions of this document appear in: Avci, N.G., Ebrahimzadeh-Pustchi, S., Akay, Y.M. et al. NF-κB inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the NF-κB(p65) and actin cytoskeleton regulatory pathways. Sci Rep 10, 13352 (2020).; and in: Pustchi, S.E.; Avci, N.G.; Akay, Y.M.; Akay, M. Astrocytes Decreased the Sensitivity of Glioblastoma Cells to Temozolomide and Bay 11-7082. Int. J. Mol. Sci. 2020, 21, 7154.