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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.

Nirmatrelvir/ritonavir association has been authorized for conditional use in the treatment of COVID-19, especially in solid-organ transplant recipients who did not respond to vaccine and are still at high risk of severe disease. This combination remains at risk of drug interactions with immunosuppressants, so monitoring drug levels seems necessary. After a simple protein precipitation of plasma sample, analytes were analyzed using an ultrahigh performance liquid chromatography system coupled with tandem mass spectrometry in a positive ionization mode. Validation procedures were based on the guidelines on bioanalytical methods issued by the European Medicine Agency. The analysis time was 4 min per run. The calibration curves were linear over the range from 10 to 1000 ng/mL for ritonavir and 40 to 4000 ng/mL for nirmatrelvir, with coefficients of correlation above 0.99 for all analytes. Intra-/interday imprecisions were below 10%. The analytical method also meets criteria of matrix effect, carryover, dilution integrity, and stability. In the context of a SARS-CoV-2 infection in a renal transplant recipient, we present a case of tacrolimus overdose with serious adverse events despite discontinuation of nirmatrelvir and ritonavir. The patient had still effective concentrations of nirmatrelvir and tacrolimus 4 days after drug discontinuation. This method was successfully applied for therapeutic drug monitoring in clinical practice.

Cancer cells in similar functional states are found in all glioblastoma, despite the genomic heterogeneity observed between and within these brain tumors. Metabolism being downstream of all signaling pathways regulating cell behaviors, we looked for metabolic weaknesses in link with motility, a key functional state for glioblastoma aggressiveness. A signature-driven data reduction approach highlighted motile cells present in thirty tumors from four independent single-cell transcriptomic datasets. Analyses integrating trajectory modeling disclosed, as characteristic of motile cells, enhanced oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). The soundness of this prediction was verified using migration and invasion assays with patient-derived cells and tissue organoids. Pharmacological and genetic manipulations showed that enhanced ROS production and MPST activity are required for the cells’ motility. Biochemical assays indicated that MPST acts by protecting protein cysteine residues from dismal hyperoxidation. In vivo, MPST knockdown translated in reduced tumor burden, and a robust increase in mice survival. These results show that enhanced oxidative stress coupled with MPST mobilization plays a key role in glioblastoma cell motility.

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.

Lactate is a central metabolite in brain physiology, involved in the astrocyte-neuron lactate shuttle, but also contributes to tumor development. Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. By adapting their glycolytic or oxidative metabolism, GBM stem-like cells are able to resist chemo- and radiotherapy. We show herein that lactate fuels GBM anaplerosis by replenishing the TCA cycle in absence of glucose. Lactate dehydrogenases (LDH) catalyze the interconversion of pyruvate and lactate. Deletion of either LDHA or LDHB did not alter significantly GBM growth and invasion. However, ablation of both LDH isoforms led to a reduction of tumor growth, and, consequently, to an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OxPhos) in the double LDHA/B KO group which sensitized tumors to cranial irradiation, massively improving mouse survival. Survival was also increased when control mice were treated with the antiangiogenic treatment, bevacizumab, and the antiepileptic drug, stiripentol which targets LDH activity. Taken together, this highlights the complex metabolic network in which both LDH A and B are integrated and underscores that combined inhibition of LDHA and B is necessary to impact tumor development. Targeting of these enzymes in combination with anti-angiogenic and repurposed drugs may be of therapeutic benefit, especially when associated with radiotherapy.

Anticancer drugs targeting angiogenesis act either directly on vascular endothelial growth factor (VEGF) receptors (VEGF inhibitors) or VEGF-mediated intracellular processes (tyrosine kinase inhibitors) or indirectly through downstream VEGF signaling within broader processes (mammalian target of rapamycin inhibitors and multiprotein kinase inhibitors). Although VEGF blockade may be implicated in arterial wall injuries, the literature on the association between antiangiogenic drugs and artery dissections or aneurysms is scant, comprising only a few case reports and 1 pharmacovigilance study conducted in Japan.1 The present study evaluated the association between all antiangiogenic drugs and dissections or aneurysms occurring in all arteries.

International audience; Glioblastoma (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.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

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. To identify potential signaling pathways involved in MT formation we performed a bioinformatics analysis of TCGA data showing that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. In particular we observed that signaling pathways involved in extracellular matrix organization are differentially expressed between these tumor entities. Using patient-derived GBM stem cell lines, we demonstrated that TGF-β1 stimulation 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. In 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.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

BACKGROUND Glioblastomas are among the most malignant primary brain tumors. GBMs are highly angiogenic, exhibit invasive growth, and elevated glycolysis. Under glycolytic conditions, glucose from the blood is metabolized in astrocytes into lactate by LDHA, and exported by MCT4 into the extracellular compartment, inducing a concomitant acidification of the microenvironment. LDHB, generally expressed in oligodendrocytes or neurons, metabolizes lactate into pyruvate for generating ATP in mitochondria. LDH expression was reported to be linked to phenotypic modifications in vitro in GBMs but the mechanisms and the precise role in vivo have not yet been investigated. MATERIAL AND METHODS We designed LDHA and LDHB Crispr-Cas9 constructs for infecting glioblastoma stem-like cells. Results: In vitro tumor cell invasion was not significantly impaired in sgLDHA glioblastoma cells, even under extreme hypoxic conditions. Tumor development was moderately impacted in terms of invasion or vascular density. We then explored the role of LDHB in these processes. LDHB knock-out cells had decreased invasive properties in vitro but surprisingly tumors were highly hemorrhagic and angiogenic, supporting a role of tumor-derived LDHB in blood vessel development. We furthermore evaluated the consequences of a double LDHA and LDHB knock-out in the glioma cells. Under hypoxic conditions, sgLDHA/B cell invasion was dramatically decreased in comparison to control cells, and apoptosis was also increased. Tumor development was dramatically impaired for LDHA/LDHB knockout tumors. CONCLUSION These results indicate the complex role of LDH enzymes in glioblastoma development. It constitutes the basis for further mechanistical studies linking lactate metabolism to brain tumor development and perturbation of the neuro-vascular microenvironment.

Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

Glioblastoma (GBM) is a common and devastating brain tumor, associated with a low median survival, despite standard therapeutic management. Among its major features, GBMs are highly angiogenic and exhibit paradoxically an elevated glycolysis. Most of differentiated cells convert glucose into pyruvate that enters into the Krebs cycle to maximize energy production in the presence of oxygen. For cancer cells, glucose uptake and catabolism are increased regardless of oxygen level. However, their energy needs are important – mainly for rapid growth – that it requires a much faster production flow. It is at this step that lactate dehydrogenase (LDH) are involved: LDHA converts efficiently pyruvate into lactate and generates NAD+ to maintain glycolysis. Thus, the lactate formed is exported into the extracellular compartment inducing an unfavourable acidification of the microenvironment. Moreover, LDHB, another LDH isoform, metabolizes lactate into pyruvate for generating energy in mitochondria. Though LDHA has already been studied in many cancers including GBM, the simultaneous role of LDH enzymes have not yet been investigated in GBM development. Hypoxia-driven LDHA expression and lactate production increased cell invasion. Infusing 13C-lactate in starved cells rescued TCA cycle. Then, we showed that, under hypoxia, double sgLDHA/B cell growth and invasion was dramatically decreased in comparison to control cells, mainly caused by an increase in apoptosis. Moreover, double impairment of LDHA and B significantly reduced tumor growth and cell invasion, and induces a massive increase in mouse survival. Tracing experiments with 13C-Glucose coupled with RNA sequencing revealed how metabolism adapts to these contraints, by modifying electron transport chain subunit expressions or by increasing lipid droplet formation. Considered for a long time as a metabolic waste, lactate is shown here to play a critical role in GBM cell symbiosis. This study highlighted GBM adaptability through the LDH isoforms and their involvement in GBM development.

Glioblastoma (GBM) is a common and devastating brain tumor, associated with a low median survival, despite standard therapeutic management. Among its major features, GBMs are highly angiogenic, infiltrative, and exhibit paradoxically an elevated glycolysis. Most of differentiated cells convert glucose into pyruvate that enters into the Krebs cycle to maximize energy production in the presence of oxygen. For cancer cells, glucose uptake and catabolism are increased regardless of oxygen level. However, tumor cell energy needs are important, mainly for rapid growth, which require a much faster production flow. Lactate dehydrogenase (LDH) are involved at this step. LDHA converts pyruvate into lactate, and generates NAD+ to maintain glycolysis. Thus, lactate is exported into the extracellular compartment inducing an acidification of the microenvironment. Moreover, LDHB, another LDH isoform, metabolizes lactate into pyruvate for generating energy in mitochondria. LDHB is generally expressed in oligodendrocytes or neurons, but also in GBM cells. Though LDHA has already been studied in many cancers including GBM, the simultaneous role of LDH enzymes have not yet been investigated in GBM development. Our results showed that hypoxia significantly increased LDHA expression and lactate production, but no changes were observed for LDHB. In presence of lactate, cell invasion was significantly increased. In vitro results showed that, under hypoxic condition, double sgLDHA/B cell growth and invasion was dramatically decreased in comparison to control cells, mainly caused by an increase in apoptosis. In vivo experiments showed that double impairment of LDHA and B significantly reduced tumor growth and cell invasion, and induces a massive increase in mouse survival. Considered for a long time as a metabolic waste, lactate is shown here to play a critical role in the tumor niche. This study highlighted GBM adaptability through the LDH isoforms and their involvement in GBM development.