Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts.

BACKGROUND: A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. METHODS: Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. RESULTS: We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood-brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. CONCLUSIONS: Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.

PKM2 diverts glycolytic flux in dependence on mitochondrial one-carbon cycle.

Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.

Magnetic resonance imaging-guided intracranial resection of glioblastoma tumors in patient-derived orthotopic xenografts leads to clinically relevant tumor recurrence.

BACKGROUND: Preclinical in vivo cancer models are essential tools for investigating tumor progression and response to treatment prior to clinical trials. Although treatment modalities are regularly assessed in mice upon tumor growth in vivo, surgical resection remains challenging, particularly in the orthotopic site. Here, we report a successful surgical resection of glioblastoma (GBM) in patient-derived orthotopic xenografts (PDOXs). METHODS: We derived a cohort of 46 GBM PDOX models that faithfully recapitulate human disease in mice. We assessed the detection and quantification of intracranial tumors using magnetic resonance imaging (MRI).To evaluate feasibility of surgical resection in PDOXs, we selected two models representing histopathological features of GBM tumors, including diffuse growth into the mouse brain. Surgical resection in the mouse brains was performed based on MRI-guided coordinates. Survival study followed by MRI and immunohistochemistry-based evaluation of recurrent tumors allowed for assessment of clinically relevant parameters. RESULTS: We demonstrate the utility of MRI for the noninvasive assessment of in vivo tumor growth, preoperative programming of resection coordinates and follow-up of tumor recurrence. We report tumor detection by MRI in 90% of GBM PDOX models (36/40), of which 55% (22/40) can be reliably quantified during tumor growth. We show that a surgical resection protocol in mice carrying diffuse primary GBM tumors in the brain leads to clinically relevant outcomes. Similar to neurosurgery in patients, we achieved a near total to complete extent of tumor resection, and mice with resected tumors presented significantly increased survival. The remaining unresected GBM cells that invaded the normal mouse brain prior to surgery regrew tumors with similar histopathological features and tumor microenvironments to the primary tumors. CONCLUSIONS: Our data positions GBM PDOXs developed in mouse brains as a valuable preclinical model for conducting therapeutic studies that involve surgical tumor resection. The high detectability of tumors by MRI across a substantial number of PDOX models in mice will allow for scalability of our approach toward specific tumor types for efficacy studies in precision medicine-oriented approaches. Additionally, these models hold promise for the development of enhanced image-guided surgery protocols.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts.

Background: A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. Methods: Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA-sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. Results: We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood-brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. Conclusions: Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice.

BACKGROUND: Numerous patient-based studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanisms behind this observation remain elusive. Our objective was to establish a preclinical model able to recapitulate this phenomenon and investigate the role of immunity underlying such protection. METHODS: An immunocompetent mouse model of allergic airway inflammation (AAI) was initiated before intracranial implantation of mouse GBM cells (GL261). RAG1-KO mice served to assess tumor growth in a model deficient for adaptive immunity. Tumor development was monitored by MRI. Microglia were isolated for functional analyses and RNA-sequencing. Peripheral as well as tumor-associated immune cells were characterized by flow cytometry. The impact of allergy-related microglial genes on patient survival was analyzed by Cox regression using publicly available datasets. RESULTS: We found that allergy establishment in mice delayed tumor engraftment in the brain and reduced tumor growth resulting in increased mouse survival. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state, uncovering a microglia gene signature, which correlated with limited local immunosuppression in glioma patients. AAI increased effector memory T-cells in the circulation as well as tumor-infiltrating CD4+ T-cells. The survival benefit conferred by AAI was lost in mice devoid of adaptive immunity. CONCLUSION: Our results demonstrate that AAI limits both tumor take and progression in mice, providing a preclinical model to study the impact of allergy on GBM susceptibility and prognosis, respectively. We identify a potentiation of local and adaptive systemic immunity, suggesting a reciprocal crosstalk that orchestrates allergy-induced immune protection against GBM.

The BET Protein Inhibitor JQ1 Decreases Hypoxia and Improves the Therapeutic Benefit of Anti-PD-1 in a High-Risk Neuroblastoma Mouse Model.

Anti-programmed death 1 (PD-1) is a revolutionary treatment for many cancers. The response to anti-PD-1 relies on several properties of tumor and immune cells, including the expression of PD-L1 and PD-1. Despite the impressive clinical benefit achieved with anti-PD-1 in several cancers in adults, the use of this therapy for high-risk neuroblastoma remains modest. Here, we evaluated the therapeutic benefit of anti-PD-1 in combination with JQ1 in a highly relevant TH-MYCN neuroblastoma transgenic mouse model. JQ1 is a small molecule inhibitor of the extra-terminal domain (BET) family of bromodomain proteins, competitively binding to bromodomains. Using several neuroblastoma cell lines in vitro, we showed that JQ1 inhibited hypoxia-dependent induction of HIF-1α and decreased the expression of the well-known HIF-1α downstream target gene CA9. Using MRI relaxometry performed on TH-MYCN tumor-bearing mice, we showed that JQ1 decreases R2* in tumors, a parameter associated with intra-tumor hypoxia in pre-clinical settings. Decreasing hypoxia by JQ1 was associated with improved blood vessel quality and integrity, as revealed by CD31 and αSMA staining on tumor sections. By analyzing the immune landscape of TH-MYCN tumors in mice, we found that JQ1 had no major impact on infiltrating immune cells into the tumor microenvironment but significantly increased the percentage of CD8+ PD-1+, conventional CD4+ PD-1+, and Treg PD-1+ cells. While anti-PD-1 monotherapy did not affect TH-MYCN tumor growth, we showed that combinatorial therapy associating JQ1 significantly decreased the tumor volume and improved the therapeutic benefit of anti-PD-1. This study provided the pre-clinical proof of concept needed to establish a new combination immunotherapy approach that may create tremendous enthusiasm for treating high-risk childhood neuroblastoma.

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency.

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.

Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted IDH1-mutant astrocytomas.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance. METHODS: Proteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice. RESULTS: We find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice. CONCLUSIONS: We show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.

Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors.

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing. For complete details on the use and execution of this protocol, please refer to Golebiewska et al. (2020).

Glioblastoma Organoids: Pre-Clinical Applications and Challenges in the Context of Immunotherapy.

Malignant brain tumors remain uniformly fatal, even with the best-to-date treatment. For Glioblastoma (GBM), the most severe form of brain cancer in adults, the median overall survival is roughly over a year. New therapeutic options are urgently needed, yet recent clinical trials in the field have been largely disappointing. This is partially due to inappropriate preclinical model systems, which do not reflect the complexity of patient tumors. Furthermore, clinically relevant patient-derived models recapitulating the immune compartment are lacking, which represents a bottleneck for adequate immunotherapy testing. Emerging 3D organoid cultures offer innovative possibilities for cancer modeling. Here, we review available GBM organoid models amenable to a large variety of pre-clinical applications including functional bioassays such as proliferation and invasion, drug screening, and the generation of patient-derived orthotopic xenografts (PDOX) for validation of biological responses in vivo. We emphasize advantages and technical challenges in establishing immunocompetent ex vivo models based on co-cultures of GBM organoids and human immune cells. The latter can be isolated either from the tumor or from patient or donor blood as peripheral blood mononuclear cells (PBMCs). We also discuss the challenges to generate GBM PDOXs based on humanized mouse models to validate efficacy of immunotherapies in vivo. A detailed characterization of such models at the cellular and molecular level is needed to understand the potential and limitations for various immune activating strategies. Increasing the availability of immunocompetent GBM models will improve research on emerging immune therapeutic approaches against aggressive brain cancer.

AN1-type zinc finger protein 3 (ZFAND3) is a transcriptional regulator that drives Glioblastoma invasion.

The infiltrative nature of Glioblastoma (GBM), the most aggressive primary brain tumor, critically prevents complete surgical resection and masks tumor cells behind the blood brain barrier reducing the efficacy of systemic treatment. Here, we use a genome-wide interference screen to determine invasion-essential genes and identify the AN1/A20 zinc finger domain containing protein 3 (ZFAND3) as a crucial driver of GBM invasion. Using patient-derived cellular models, we show that loss of ZFAND3 hampers the invasive capacity of GBM, whereas ZFAND3 overexpression increases motility in cells that were initially not invasive. At the mechanistic level, we find that ZFAND3 activity requires nuclear localization and integral zinc-finger domains. Our findings indicate that ZFAND3 acts within a nuclear protein complex to activate gene transcription and regulates the promoter of invasion-related genes such as COL6A2, FN1, and NRCAM. Further investigation in ZFAND3 function in GBM and other invasive cancers is warranted.

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Altered metabolic landscape in IDH-mutant gliomas affects phospholipid, energy, and oxidative stress pathways.

Heterozygous mutations in NADP-dependent isocitrate dehydrogenases (IDH) define the large majority of diffuse gliomas and are associated with hypermethylation of DNA and chromatin. The metabolic dysregulations imposed by these mutations, whether dependent or not on the oncometabolite D-2-hydroxyglutarate (D2HG), are less well understood. Here, we applied mass spectrometry imaging on intracranial patient-derived xenografts of IDH-mutant versus IDH wild-type glioma to profile the distribution of metabolites at high anatomical resolution in situ This approach was complemented by in vivo tracing of labeled nutrients followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Selected metabolites were verified on clinical specimen. Our data identify remarkable differences in the phospholipid composition of gliomas harboring the IDH1 mutation. Moreover, we show that these tumors are characterized by reduced glucose turnover and a lower energy potential, correlating with their reduced aggressivity. Despite these differences, our data also show that D2HG overproduction does not result in a global aberration of the central carbon metabolism, indicating strong adaptive mechanisms at hand. Intriguingly, D2HG shows no quantitatively important glucose-derived label in IDH-mutant tumors, which suggests that the synthesis of this oncometabolite may rely on alternative carbon sources. Despite a reduction in NADPH, glutathione levels are maintained. We found that genes coding for key enzymes in de novo glutathione synthesis are highly expressed in IDH-mutant gliomas and the expression of cystathionine-ß-synthase (CBS) correlates with patient survival in the oligodendroglial subtype. This study provides a detailed and clinically relevant insight into the in vivo metabolism of IDH1-mutant gliomas and points to novel metabolic vulnerabilities in these tumors.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

BACKGROUND: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. METHODS: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. RESULTS: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. CONCLUSIONS: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

Aptamer Functionalization of Nanosystems for Glioblastoma Targeting through the Blood-Brain Barrier.

Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Gint4.T aptamer specifically recognizes platelet-derived growth factor receptor ß and can cross the blood-brain barrier (BBB). We synthesized Gint4.T-conjugated PNPs able of high uptake into U87MG glioblastoma (GBM) cells and with astonishing EC50 value (38 pM) when loaded with a PI3K-mTOR inhibitor. We also demonstrated in vivo BBB passage and tumor accumulation in a GBM orthotopic model.

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma.

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFß1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.

CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis.

A critical process underlying cancer metastasis is the acquisition by tumor cells of an invasive phenotype. At the subcellular level, invasion is facilitated by actin-rich protrusions termed invadopodia, which direct extracellular matrix (ECM) degradation. Here, we report the identification of a new cytoskeletal component of breast cancer cell invadopodia, namely cysteine-rich protein 2 (CRP2). We found that CRP2 was not or only weakly expressed in epithelial breast cancer cells whereas it was up-regulated in mesenchymal/invasive breast cancer cells. In addition, high expression of the CRP2 encoding gene CSRP2 was associated with significantly increased risk of metastasis in basal-like breast cancer patients. CRP2 knockdown significantly reduced the invasive potential of aggressive breast cancer cells, whereas it did not impair 2D cell migration. In keeping with this, CRP2-depleted breast cancer cells exhibited a reduced capacity to promote ECM degradation, and to secrete and express MMP-9, a matrix metalloproteinase repeatedly associated with cancer progression and metastasis. In turn, ectopic expression of CRP2 in weakly invasive cells was sufficient to stimulate cell invasion. Both GFP-fused and endogenous CRP2 localized to the extended actin core of invadopodia, a structure primarily made of actin bundles. Purified recombinant CRP2 autonomously crosslinked actin filaments into thick bundles, suggesting that CRP2 contributes to the formation/maintenance of the actin core. Finally, CRP2 depletion significantly reduced the incidence of lung metastatic lesions in two xenograft mouse models of breast cancer. Collectively, our data identify CRP2 as a new cytoskeletal component of invadopodia that critically promotes breast cancer cell invasion and metastasis.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.