Glioblastoma multiforme (GBM) is the deadliest form of primary brain tumor. GBM tumors are highly heterogeneous, being composed of tumor cells as well as glioblastoma stem cells (GSCs) that contribute to drug resistance and tumor recurrence following treatment. To develop therapeutic strategies, an improved understanding of GSC behavior in their microenvironment is critical. Herein, we have employed three‐dimensional (3D) hyaluronic acid (HA) hydrogels that allow the incorporation of brain microenvironmental cues to investigate GSC behavior. U87 cell line and patient‐derived D456 cells were cultured as suspension cultures (serum‐free) and adherently (in the presence of serum) and were then encapsulated in HA hydrogels. We observed that all the seeded single cells expanded and formed spheres, and the size of the spheres increased with time. Increasing the initial cell seeding density of cells influenced the sphere size distribution. Interestingly, clonal expansion of serum‐free grown tumor cells in HA hydrogels was observed. Also, stemness marker expression of serum and/or serum‐free grown cells was altered when cultured in HA hydrogels. Finally, we demonstrated that HA hydrogels can support long‐term GSC culture (up to 60 days) with retention of stemness markers. Overall, such biomimetic culture systems could further our understanding of the microenvironmental regulation of GSC phenotypes.
Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. The GBM microenvironment is highly heterogeneous and is composed of many cell types including astrocytes and endothelial cells (ECs) along with tumor cells, which are responsible for heightened resistance to standard chemotherapeutic drugs such as Temozolomide (TMZ). Here, we investigated how drug treatments impact stemness marker expression of GBM cells in multicellular tumor spheroid (MCTS) models. Co‐ and tri‐culture MCTS constructed using U87‐MG GBM cells, astrocytes, and/or ECs were cultured for 7 days. At Day 7, 5 μM lonafarnib (LNF), 100 μM TMZ, or combination of 5 μM LNF + 100 μM TMZ was added and the MCTS were cultured for an additional 48 h. We assessed the spheroid sizes and expression of stemness markers‐ NESTIN, SOX2, CD133, NANOG, and OCT4‐ through qRT‐PCR and immunostaining. Following 48 h treatment with LNF, TMZ or their combination (LNF + TMZ), the spheroid sizes decreased compared to the untreated control. We also observed that the expression of most of the stemness markers significantly increased in the LNF + TMZ treated condition as compared to the untreated condition. These results indicate that drug treatment can influence the stemness marker expression of GBM cells in MCTS models and these aspects must be considered while evaluating therapies. In future, by incorporating other relevant cell types, we can further our understanding of their crosstalk, eventually leading to the development of new therapeutic strategies.
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