The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids

Abstract

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.

Matrix stiffness influences response to chemo and targeted therapy in brain metastatic breast cancer cells

“…After 48 h treatment, the trypsinized cells were washed with 1× PBS and RNA was extracted and transcribed into cDNA using GeneJET RNA Purification Kit (Thermo Scientific) as previously described. 26 Quantitative RT-PCR (Applied Biosystems StepOnePlus Real-time PCR System) was used to quantify gene expression using the SYBR Green Master Mix. The GAPDH (housekeeping) gene was used to compare the relative expression of SGK1 gene by ΔΔCt method where ΔCt = ΔCt, gene of interest (SGK1) −ΔCt, GAPDH .…”

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells

“…To qualitatively assess cell viability, we stained cells with Calcein AM (Thermo Fisher Scientific) as described previously. 24 Briefly, on day 5, cell seeded hydrogels were incubated in 100 μL of fresh media containing 4 Â 10 À6 M Calcein AM for 1 h at 37 C and 5% CO 2 . The samples were then washed in PBS (Gibco).…”

SOX2 in Glioblastoma: Regulation Mechanisms and A Potential Therapeutic Target

“…However, SOX2 is being used directly or indirectly by several strategies to combat GSCs. The GBM microenvironment is highly heterogeneous and consists of many cell types including astrocytes and endothelial cells as well as tumor cells, which are responsible for increased resistance to standard chemotherapeutic agents such as TMZ and radiotherapy [116]. Therefore, it is of great importance to study how drug treatment affects the expression of GBM cell lineage markers in multicellular tumor (MCTS) models.…”