Speaker
Description
Introduction
A deeper insight into the tumor microenvironment (TME) is crucial for advancing cancer research. In order to study the TME in depth, 3D cell culture models are preferred over traditional 2D cultures, as they offer a more accurate representation of cellular behaviour and functional aspects, when interacting with an extracellular matrix (ECM) like structure. This includes key processes such as proliferation, adhesion, and migration [1]. In 3D cultures, hydrogels are commonly used to mimic the ECM. In order to investigate brain cancers, like glioblastoma, the stiffness of the hydrogel must be low to simulate the human brain tissue in vivo, which typically ranges between 50–200 Pa depending on the region and development [2].
Methods
Two human glioblastoma cell lines (U87-MG and LN18) were cultivated in five commonly used pure hydrogels: collagen, alginate, thiolated hyaluronic acid (HA-SH), oxidized alginate-gelatin (ADA-Gel), and Matrigel. The hydrogels were measured for stiffness by cyclic compression tests up to a 15 % strain. Additionally, the cells were evaluated for their morphology, viability, senescence, as well as gene expression, with a focus on inflammatory markers.
Results
Conducting cyclic compression tests, we demonstrate that these hydrogels displayed shear moduli between 30-110 Pa, similar to slices of native human brains. Both glioblastoma cell lines exhibited a more astrocytic-like morphology in collagen and Matrigel, characterized by spread-out cells with protrusions. In contrast, cells in HA-SH, ADA-Gel, and alginate showed a more rounded spheroid-like morphology without notable outgrowth. This morphological trend was consistent with viability data, with cells showing the highest viability in collagen and Matrigel after six days of cultivation. Establishing a 3D senescence assay (3D-X-GAL), we demonstrated that both cell lines in alginate, ADA-Gel, collagen have a higher cellular β-galactosidase activity than in Matrigel and HA-SH. Additionally, the cells displayed hydrogel-induced inflammatory gene expressions, like IL-6, TNFA and the cytokines CXCL9 and CXCL10 compared to 2D cell cultures.
Discussion
These findings highlight the importance of hydrogel selection for the outcome of 3D culture models. The physical and biochemical properties of the matrix significantly influence cell behaviour. In addition, depending on the hydrogel used differences of a cell cycle arrest or senescence as well as expression of inflammatory genes were found. It should be further evaluated, if the hydrogels cause an inflammation induced senescence or a senescence-associated secretory phenotype (SASP). To better mimic the brain TME, we are currently expanding multiple hydrogel materials and incorporating additional brain-specific components, such as anchoring proteins (e.g. fibronectin, laminin, brevican) or lipids [3,4,]. Co-culturing glioblastoma cells with other brain cell types, such as neurons, astrocytes, or microglia, may also enhance the physiological relevance of the model.
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