6 research outputs found
TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1
International audienceAbstract Background Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. Methods Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-β) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β. Results Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. Conclusion TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network
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Abstract 331: Mitochondrial transfer from astrocytes drives glioblastoma tumorigenicity
Abstract Mitochondrial transfer in the central nervous system occurs from astrocytes to neurons in stroke. Mitochondrial exchange has also been reported among tumor cells in glioblastoma (GBM), the most common primary brain tumor. However, the role of mitochondrial transfer from non-neoplastic cells in the surrounding microenvironment to GBM remains poorly understood. We hypothesized that mitochondrial transfer from these non-neoplastic to GBM cells supports tumor metabolism and growth. Using transgenic mice expressing fluorophore-tagged mitochondria, we found that ~50% of orthotopically-implanted mouse GBM cells acquire host mitochondria. Brain-resident cells, mainly astrocytes, but not infiltrating immune cells were the primary mitochondrial donors in vivo and in vitro. Mitochondrial transfer also occurred from immortalized human astrocytes to a broad array of patient-derived xenograft (PDX) models of GBM in vitro at rates of 15-35%. GBM cells that acquired mitochondria expressed higher levels of the ATP-synthase subunit ATP5A and produced more ATP, while metabolomics revealed upregulated amino acid metabolism in recipient cells. In vivo, mouse GBM cells that acquired mitochondria were more likely to be in G2/M proliferative cell cycle phases. We observed a similar effect in PDX that acquired astrocyte mitochondria from co-cultures in vitro. To mechanistically link increased proliferation specifically to mitochondrial transfer, we isolated astrocyte mitochondria by differential centrifugation and found that addition and uptake of cell-free mitochondria in human GBM cells recapitulated the increased proliferation. Using sorted mouse and human GBM cells with/without astrocyte mitochondrial acquisition, we further found that mitochondrial transfer promoted in vitro self-renewal and in vivo tumorigenicity, leading to significant reduction in survival and increased penetrance in orthotopic GBM models. Transfer in mouse and human systems was contact-dependent and was abrogated by physical separation of donor and recipient cells by transwell inserts. We visualized contact-dependent transfer across actin-based intercellular connections consistent with previously reported microtubes. We confirmed the critical role of actin and the actin-associated protein, growth-associated protein 43 (GAP43) in facilitating mitochondrial transfer by showing that pharmacologic inhibition and genetic knockdown (respectively) significantly decreased the rate of mitochondrial transfer. Taken together, mitochondrial transfer comprises a fundamental, protumorigenic mechanism of GBM, enhancing metabolic activity and driving tumor cell proliferation. Further elucidating the molecular machinery regulating astrocyte mitochondrial transfer and its downstream protumorigenic effects will lead to therapeutic opportunities targeting this understudied tumor microenvironment interaction. Citation Format: Dionysios C. Watson, Defne Bayik, Simon Storevik, Shannon S. Moreino, Samuel S. Sprowls, Gauravi Deshpande, Palavalasa Sravya, Costas A. Lyssiotis, Daniel R. Wahl, Hrvoje Miletic, Justin D. Lathia. Mitochondrial transfer from astrocytes drives glioblastoma tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 331
Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development
International audienceGlioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12-15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model
TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1
BACKGROUND: Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. METHODS: Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate microtube formation under TGF-βstimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β. RESULTS: Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF- β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. CONCLUSION: TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network
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CNSC-15. MITOCHONDRIA TRANSFER VIA GLIOMA-ASTROCYTE NETWORK MICROTUBES REPROGRAMS TUMOR CELLS FOR ENHANCED TUMORIGENICITY
Abstract Glioblastoma (GBM) interaction with neural cells is critical to its pathobiology. Emerging evidence suggests that GBM cells form an interconnected network with astrocytes, facilitating tumor persistence. Given reports of intercellular transfer of mitochondria in ischemic stroke and other pathologic disease states outside the CNS, we hypothesized that this network facilitates mitochondria transfer from astrocytes to GBM with protumorigenic sequelae. Employing transgenic mice and intracranial viral vector transductions in rats, we found that mitochondria transfer from the TME to GBM occurs in intracranial mouse and patient-derived xenograft models (in nude rats) of GBM. Mitochondria transfer from bone marrow-derived immune cells was minimal in bone marrow chimera mouse models of orthotopic GBM, suggesting that neural cells were the primary mitochondria donors. We confirmed this in vitro, where mouse astrocytes were the major mitochondria donors, followed by microglia and to a much smaller extent bone marrow-derived macrophages. Immortalized human astrocytes transduced with mitochondria-localized mCherry (mito-mCherry) also transferred their mitochondria to numerous patient-derived glioma stem cell (GSC) models at rates of ~5-20%, assessed by flow cytometry and confocal microscopy. Mitochondria were visualized along intercellular actin bridges, structurally resembling tumor microtubes. Blocking actin polymerization or knocking down GAP43 (previously linked to microtube formation) decreased mitochondria transfer from astrocytes to GBM in vitro. Functionally, sorted mito-mCherry+ patient-derived GSCs displayed higher mitochondrial respiration, metabolomic reprogramming and proliferation-promoting phospho-signaling. Mito-mCherry+ GBM cells were more likely to be in the proliferative G2/M phases of the cell cycle, and when sorted from co-cultures had high self-renewal (in vitro) and tumor-initiating capacity (in vivo xenograft mouse model). In ongoing work, we are investigating the role of retrograde GBM to astrocyte transfer of mitochondria by dual-color labeling of the organelle, as well as further delineating the protein machinery involved in this fundamental protumorigenic process, with the goal of identifying novel therapeutic targets
GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity
The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development