TMIC-93. TGF-β facilitated mitochondria transfer in glioblastoma enhances tumor invasion and formate overflow

Abstract BACKGROUND The acquisition of intact mitochondria from the tumor microenvironment (TME) is a widespread phenomenon in glioblastoma multiforme (GB), supporting mitochondrial respiration and metabolic pathways linked to proliferation and tumorigenicity¹. The Transforming Growth Factor β (TGF-β) signaling pathway is significantly upregulated in GB compared to lower-grade gliomas² and is implicated in the formation of microtubes (MTs)³. We hypothesized that TGF-β impacts mitochondrial transfer between GB cells and components of the TME, particularly astrocytes. We investigated if the transfer enhances tumor invasiveness and invasion associated formate overflow¹. Additionally, we investigated whether metabolic stress via mitochondrial depletion would influence mitochondrial transfer at the GB–TME interface. METHODOLOGY We used human GB cell lines to investigate mitochondria transfer and microtube formation by confocal microscopy and flow cytometry under TGF-β and/or TGF- β receptor/downstream inhibitors. Invasive potential was assessed using collagen-based and ex vivo organoid invasion assays, alongside quantification of formate release. Mitochondrial depletion was induced using the Parkin-YFP/CCCP model4. For in vivo studies, orthotopic xenograft models were used to evaluate the impact of mitochondrial depletion, and immunohistochemistry was performed to quantify mitochondrial transfer. RESULTS Here we show that TGF-β treatment of GB–astrocyte co-cultures significantly enhances mitochondria transfer rates and MT formation, which can be blocked by Galunisertib or SMAD inhibitors. GB cells acquiring mitochondria from the tumor microenvironment exhibited increased invasiveness and elevated formate production. In a mitochondrial depletion model, GBM cells demonstrated enhanced uptake of mitochondria from the microenvironment, highlighting their reliance on this process to support aggressive tumor growth. CONCLUSION This study identifies a regulatory role for TGF-β in glioblastoma–TME interactions, with SMAD signaling as a key pathway. Mitochondrial transfer from astrocytes supports GB proliferation, invasion, and survival. REFLECTION Our findings offer new insights into communication of GB with its microenvironment, which may uncover novel treatment opportunities.