Horizontal mitochondria transfer stimulates pro-tumorigenic astrocyte reprogramming in glioblastoma

Glioblastoma (GBM), the most aggressive primary brain tumor, co-opts astrocytes in the tumor microenvironment (TME) to facilitate growth, immunosuppression, and therapeutic resistance. However, the signaling mechanisms driving these pro-tumorigenic astrocyte states are poorly defined. We previously showed that astrocytes donate mitochondria to GBM cells to augment GBM metabolism and self-renewal, but the role of mitochondria transfer in astrocyte biology and TME signaling remains unclear. To assess mitochondria transfer to astrocytes, we transduced patient-derived GBM cells (PDCs) with mitochondria-localized GFP (mito-GFP) and co-cultured them with human astrocytes, revealing that GBM cells transfer mitochondria to astrocytes in a cell type-dependent manner, with rates approaching 40% in some models. To additionally validate transfer, we performed deep mitochondrial DNA (mtDNA) sequencing of co-cultured astrocytes and detected transfer of unique PDC mtDNA variants to astrocytes. To evaluate transfer in the tumor microenvironment, we intracranially implanted mito-GFP PDCs in an orthotopic mouse model and found that tumor-associated astrocytes contained GBM-derived mitochondria at similar frequencies to in-vitro observations. Initial survival studies demonstrated that co-implantation of astrocytes with PDCs increased tumorigenesis, and we subsequently showed that co-implantation of astrocytes harboring GBM mitochondria further accelerated tumor growth. To characterize the functional consequences of GBM mitochondria transfer, we assessed transfer-positive astrocyte phenotypes and observed increased cell cycling, proliferation, cellular ROS, and altered oxidative metabolism. Critically, we found that astrocytes acquiring GBM mitochondria increased secretion of the chemokine CCL2 and facilitated enhanced recruitment of immunosuppressive myeloid cells. Collectively, these results suggest that GBM mitochondria transfer to astrocytes is both a pro-tumorigenic mechanism and a therapeutic vulnerability in the GBM TME. Ongoing studies will define the astrocyte signaling networks activated by mitochondria acquisition and identify molecular regulators of transfer initiation to enable therapeutic intervention.