Glioblastoma mechanobiology at multiple length scales.

Glioblastoma multiforme (GBM), a primary brain cancer, is one of the most aggressive forms of human cancer, with a very low patient survival rate. A characteristic feature of GBM is the diffuse infiltration of tumor cells into the surrounding brain extracellular matrix (ECM) that provide biophysical, topographical, and biochemical cues. In particular, ECM stiffness and composition is known to play a key role in controlling various GBM cell behaviors including proliferation, migration, invasion, as well as the stem-like state and response to chemotherapies. In this review, we discuss the mechanical characteristics of the GBM microenvironment at multiple length scales, and how biomaterial scaffolds such as polymeric hydrogels, and fibers, as well as microfluidic chip-based platforms have been employed as tissue mimetic models to study GBM mechanobiology. We also highlight how such tissue mimetic models can impact the field of GBM mechanobiology.

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

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.

The Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids.

INTRODUCTION: 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 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, which has not been previously investigated. METHODS: We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining. RESULTS: We incorporated 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. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers. CONCLUSIONS: We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00691-y.

Three-dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors.

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.

Biomimetic models to examine microenvironmental regulation of glioblastoma stem cells.

Glioblastoma multiforme (GBM), a malignant brain tumor, is the deadliest form of human cancer with low survival rates because of its highly invasive nature. In recent years, there has been a growing appreciation for the role that glioblastoma stem cells (GSCs) play during tumorigenesis and tumor recurrence of GBM. GSCs are a specialized subset of GBM cells with stem cell-like features that contribute to tumor initiation and therapeutic resistance. Thus, to enhance therapeutic efficiency and improve survival, targeting GSCs and their microenvironmental niche appears to be a promising approach. To develop this approach, understanding GSC-microenvironment interactions is crucial. This review discusses various biomimetic model systems to understand the impact of biophysical, biochemical, and cellular microenvironmental cues on GSC behaviors. These models include two-dimensional or matrix-free environment models, engineered biomaterial-based three-dimensional models, co-culture models, and mouse and rat in vivo models. These systems have been used to study the effects of biophysical factors, modulation of signaling pathways, extracellular matrix components, and culture conditions on the GSC phenotype. The advantages and disadvantages of these model systems and their impact in the field of GSC research are discussed.