The ability of glioma cells to invade adjacent tissues is a key hallmark of tumour progression and a major determinant of patient outcome. Due to the high tumour heterogeneity and invasiveness of high-grade gliomas (Glioblastoma - GB), the current therapies remain largely ineffective, highlighting the need for novel therapeutic approaches. The Transmembrane BAX Inhibitor Motif Containing (TMBIM) protein family, consisting of six intracellular ion channels, is implicated in multiple cell processes associated with cancer hallmarks. The Golgi-localized TMBIM4 regulates intracellular Ca²⁺ fluxes, apoptosis, and cell motility. Here, we investigate the role of TMBIM4 in glioma cell invasion and metabolic adaptation.

Glioma transcriptomic datasets from TCGA, CGGA, Rembrandt, and Ivy_GAP were analysed to determine TMBIM1-6 mRNA levels and their association with tumour grade and patient survival. The cellular role of TMBIM4 was assessed in human glioblastoma U87 and U251 cells following TMBIM4 KD using siRNA. Cell invasion was measured in 2D and 3D cultures, and an orthotopic mouse model was used to analyse tumour growth/invasion. Lipid peroxidation, DNA damage, and Cystathionine Beta-Synthase levels were evaluated by immunofluorescence. Quantitative mass spectrometry was used to assess protein expression.

TMBIM1, 4, and 6 mRNA were significantly upregulated in gliomas, particularly in GB. Their expression correlated with tumour grade and was associated with reduced patient survival, supporting their potential prognostic value. Interestingly, TMBIM4 exhibited elevated expression in hyperplastic blood vessels and regions of microvascular proliferation, hinting at its involvement in tumour invasion or angiogenesis.TMBIM4 KD significantly reduced GB cell invasion in both 2D and 3D models without affecting cell viability or proliferation and led to a marked reduction in tumour growth in vivo. Lower expression of TMBIM4 increased lipid peroxidation and DNA damage, indicating a role in oxidative stress resistance. Proteomic analysis further uncovered a reprogramming of amino acid and lipid synthesis-related proteins upon TMBIM4 KD. A decreased expression of CBS, a key enzyme in cysteine biosynthesis, was detected in the tumour cells in vivo. Supplementation of cell media with glutathione (GSH) rescued the 2D invasive phenotype of TMBIM4 KD cells, suggesting a role for GSH in TMBIM4-dependent tumour invasion.

Our findings suggest that TMBIM4 promotes glioma metabolic reprogramming, contributing to redox homeostasis and cell invasion. Targeting TMBIM4 could represent a novel therapeutic strategy to reduce glioma progression. Acknowledgments: FCT (UIDB04567/2020, UIDP/04567/2020, UI/BD/151424/2021), SeedFunding (COFAC/ILIND/CBIOS/2/2024) and COST (CA15214-47282).