P12.58.a - Role of Il-33/ST2 axis in glioblastoma development and its response to treatment

BACKGROUND Glioblastoma (GBM) is the most aggressive and lethal primary brain tumour, characterised by rapid proliferation, extensive migration, and resistance to therapies driven by stem-like GBM cells. Inflammatory pathways may play a critical role in modulating GBM migration and therapeutic resistance. The interleukin-33 (IL-33)/ST2 axis has garnered attention for activating downstream pathways that promote cell survival, growth, migration, and invasion. This study aims to explore the role of IL-33/ST2 signalling in GBM cell proliferation, migration, stemness, and treatment response, highlighting its potential contribution to GBM aggressiveness. MATERIAL AND METHODS The effects of IL-33 supplementation (5, 10, 20, and 50 ng/mL) on cell proliferation were evaluated in 2D (cell viability assay) and 3D (tumour spheroid) models in 4 patient-derived GBM stem cell lines (G7, E2, OX5-core, OX5-edge) cultured in stem cell-enriched conditions. Migration was assessed via wound healing and transwell assays. GBM-stem cell frequency was measured using a 3D extreme limiting dilution assay. IL-33, ST2, and γH2AX expression post-irradiation (0 and 2 Gy) were analysed by immunofluorescence to assess IL-33/ST2 axis responses to DNA damage. Statistical analysis was performed using unpaired t test or two-way ANOVA with appropriate post hoc tests, and ELDA software for stem cell frequency estimation. RESULTS IL-33 treatment significantly enhanced the migratory capacity of all four GBM cell lines in a concentration-dependent manner in wound healing assays, with 20 ng/mL exhibiting a 2.30(p≤0.01), 1.18(p≤0.01), 2.31(p≤0.0001), 1.75(p≤0.001) fold increase in G7, E2, OX5(core) and OX5(edge), respectively, compared to vehicle. This pro-migratory effect was further confirmed using Transwell assays, exhibiting a 1.84(p≤0.0001), 2.15(p≤0.0001), 5.14(p≤0.0001) and 1.22(p≤0.0001) fold in G7, E2, OX5(core) and OX5(edge) cells separately, treated with IL-33 compared to vehicle, suggesting a pro-migratory role of IL-33 in GBM. Under 2D or 3D conditions, IL-33 did not impact GBM cell proliferation or stemness. ST2 cytoplasmic expression in G7 GSCs was significantly increased (p≤0.01) following IR treatment, indicating that radiation may modulate IL-33/ST2 activity in GBM cells across the examined 24h time-course. However, no colocalisation was observed between IL-33, ST2 and γH2AX expression, suggesting spatial separation of IL-33/ST2 signalling from DNA damage response foci. CONCLUSION Our study provides novel insights into the role of the IL-33/ST2 axis in GBM progression, highlighting its ability to enhance cell migration while having minimal impact on proliferation and stemness. The modulation of ST2 expression following RT suggests that IL-33/ST2 axis may influence GBM responses to radiation, warranting further investigation into its role in radiation resistance.