J4 - Matrix mechanics drive changes in polyamine metabolism in glioblastoma across different oxygen levels

Introduction: : Glioblastoma (GBM) is a lethal brain cancer with a median survival time of less than 15 months. GBM tumors have a heterogeneous microenvironment generally characterized by a necrotic core surrounded by a dense, hypoxic bulk that is stiffer than the brain parenchyma into which the cancer cells invade. The tumor edge has more functional microvessels, and thus more normoxic oxygen levels, although heterogeneity is still present. This heterogeneity of matrix stiffness and oxygen levels creates distinct biophysical niches within the tumor that promote growth and survival of GBM cells. However, few studies have investigated the overlapping effects of these biophysical features on GBM aggression. Previously, we identified matrix-driven changes in glucose metabolism, where soft, brain-like matrices (G’=100 Pa) promoted glycolysis and stiff, tumor-like matrices (G’=1000 Pa) promoted oxidative metabolism (Sohrabi, et al. Cell Reports 2023). These metabolic changes also correlated with differences in cellular invasion in proliferation. Thus, in this study we expanded on this work to investigate metabolic and phenotypic changes in GBM cells cultured in biomimetic matrices with distinct elasticity and at different oxygen levels.

Materials and

Methods: : Patient-derived GBM cells were grown into uniform spheroids using AggreWellTM microwell plates and then embedded into hyaluronic acid-based hydrogels with storage moduli of 100 Pa, to mimic noncancerous brain tissue (soft), or 1000 Pa, to mimic the tumor bulk (stiff). These 3D culture systems were then incubated in 21% or 1% O2 for 7 days. Bioluminescence readings via firefly luciferase and brightfield images were taken over time to monitor cell growth and invasion, respectively. On day 7, cells were extracted from the hydrogel for LC-MS metabolomics and EdU assay was used to quantify cell proliferation. Western blot was used to measure expression and activation of metabolic enzymes and downstream proteins of interest.

Results, Conclusions, and Discussions:: LC-MS metabolomics revealed distinct alterations in several metabolic pathways driven largely by matrix stiffness. Polyamine metabolism, which has been shown to play a role in cell proliferation, invasion, and treatment resistance, is significantly altered by matrix stiffness in multiple patient cell lines. We observed higher levels of polyamines in soft matrices compared to stiff matrices in both 21% and 1% O2. Downstream of polyamine metabolism, the polyamine spermidine is used to hypusinate, and therefore activate, eukaryotic translation initiation factor 5A (EIF5A), which is important for the efficient translation of intracellular proteins involved in proliferation, cytoskeleton remodeling, and mitochondrial function. Western blot revealed higher levels of activated EIF5A in stiff matrices than soft matrices, particularly in 1% O2, which suggests that reduced polyamine levels in stiff matrices may be at least in part due to usage downstream for EIF5A hypusination. In line with known effects of EIF5A on cell proliferation, we have also observed increased proliferation in stiff matrices. The HK177 patient cell line proliferates more in stiff matrices in both 21% O2 and 1% O2, while the HK408 patient line only has increased proliferation in stiff conditions in 1% O2. These findings reveal that matrix stiffness plays an important role in metabolic regulation even in low oxygen conditions. They also begin to explore how stiffness-mediated changes in polyamine metabolism may drive phenotypic changes in varying biophysical niches within the GBM tumor.

Acknowledgements and/or References (Optional)::