Poster T8 - Overlapping contributions of matrix mechanics and hypoxia on glioblastoma phenotype, phosphosignaling, and metabolism

Introduction: Glioblastoma (GBM) is a uniformly lethal brain cancer due to poor treatment response and high rates of recurrence. The unique brain environment is believed to play a key role in GBM progression and malignancy, as GBMs very rarely metastasize to other organs. Despite promising pre-clinical results of several treatments in the past twenty years, the standard of care has not changed since the addition of concomitant temozolomide in 2005 due to unsuccessful clinical trials (Stupp, et al. N Engl J Med 2005, Angom, et al. Brain Sci 2023). This may be at least partially due to a lack of biomimetic ex vivo models that simulate the complex microenvironmental influences present in GBMs in vivo. Previously, we characterized the effect of varying matrix stiffness on GBM cell proliferation, invasion, and metabolism in 3D hydrogels mimicking the stiffness of noncancerous brain tissue (G’ = 100 Pa) or the tumor bulk (G’ = 1000 Pa) (Sohrabi, et al. Cell Reports 2023). While these observations were made using cultures maintained at 21% O2, the O2 concentration in brain tissue is lower and hypoxia is known to affect cell metabolism and GBM progression. Thus, we cultured GBM spheroids in 3D matrices with defined elastic moduli in 21% or 1% O2 to identify how these effects interact to determine GBM behavior. Although the influences of matrix stiffness and hypoxia on GBM cell metabolism and phenotypic behavior likely have overlapping effects, they have yet to be studied with variable decoupling to elucidate the contributions of each factor.

Materials and

Methods: Patient-derived GBM cells were grown into spheroids and then encapsulated in hyaluronic acid-polyethylene glycol hydrogels with storage moduli of 100 Pa (soft matrices) or 1000 Pa (stiff matrices) to match the stiffness of noncancerous brain tissue and the GBM tumor bulk, respectively, and grown in either 21% or 1% O2. Bioluminescence imaging of constitutively expressed firefly luciferase was used to estimate cell numbers, while cell invasion was tracked over time using brightfield imaging. Phosphosignaling was analyzed using Luminex multiplexed immunoassays to quantify total expression and phosphorylation of prominent oncogenic signaling pathways. Fluorescence lifetime imaging microscopy (FLIM) was used to assess NADH state as a metabolic signature in addition to LC-MS metabolomics. Immunofluorescent staining was used to measure expression of proteins of interest such as HIF1α, cl-PARP, and Ki67.

Results, Conclusions, and Discussions: Cl-PARP immunostaining revealed that GBM cells grown in 1% O2 are more apoptotic than those grown in 21% O2 regardless of matrix stiffness. In line with previous findings (Sohrabi, et al. Cell Reports 2023), GBM cells cultured in soft matrices migrated significantly more than cells cultured in stiff conditions, regardless of O2 levels. However, GBM cells derived from multiple patient tumors and cultured in soft matrices were found to be more invasive in 1% O2 than in 21% O2. GBM cells cultured in soft or stiff matrices in 1% or 21% O2 displayed distinct activities in phosphosignaling pathways, including Ras/Raf/MAPK, Src, and Akt, over 7 days in culture. After 24 hrs in culture, cells in stiff matrices cultured in either 1% or 21% O2 had increased activity across proliferation and survival pathways (Figure 1A); however, as GBM spheroids became more hypoxic in 1% O2 conditions over time, these cells began to downregulate proliferation-related signaling pathways (Figure 1B-C). An exception was the Src pathway, whose signaling remained consistently upregulated in stiff matrix conditions over time regardless of oxygen condition. Principal components analysis (PCA) of phosphosignaling data revealed larger variance between oxygen levels in stiff conditions compared to soft conditions. LC-MS metabolomics similarly identified distinct trends in metabolite production associated with stiffness and oxygen conditions. Synthesis of polyamines, in particular spermine and spermidine, was increased in cells cultured in soft matrices compared to stiff matrices in both oxygen conditions. Results demonstrate both distinct and overlapping effects of oxygen levels and tissue stiffness on GBM cell phenotype, signaling, and metabolism using a biomimetic 3D culture system and substantiate the need for further studies to elucidate the specific effects of matrix stiffness and oxygen levels on GBM malignancy.

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