Microglia promote glioblastoma via mTOR‐mediated immunosuppression of the tumour microenvironment

Article22 June 2020Open Access Source DataTransparent process Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment Anaelle A Dumas Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Nicola Pomella Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Gabriel Rosser Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Loredana Guglielmi Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Claire Vinel Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Thomas O Millner Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Jeremy Rees National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Natasha Aley Division of Neuropathology, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Denise Sheer orcid.org/0000-0001-9067-1796 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Jun Wei Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Anantha Marisetty Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Amy B Heimberger Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Robert L Bowman Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Sebastian Brandner orcid.org/0000-0002-9821-0342 National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Johanna A Joyce Department of Oncology, Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Silvia Marino Corresponding Author [email protected] orcid.org/0000-0002-9612-2883 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Anaelle A Dumas Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Nicola Pomella Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Gabriel Rosser Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Loredana Guglielmi Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Claire Vinel Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Thomas O Millner Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Jeremy Rees National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Natasha Aley Division of Neuropathology, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Denise Sheer orcid.org/0000-0001-9067-1796 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Jun Wei Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Anantha Marisetty Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Amy B Heimberger Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Robert L Bowman Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Sebastian Brandner orcid.org/0000-0002-9821-0342 National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK Search for more papers by this author Johanna A Joyce Department of Oncology, Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Silvia Marino Corresponding Author [email protected] orcid.org/0000-0002-9612-2883 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK Search for more papers by this author Author Information Anaelle A Dumas1, Nicola Pomella1, Gabriel Rosser1, Loredana Guglielmi1, Claire Vinel1, Thomas O Millner1, Jeremy Rees2, Natasha Aley3, Denise Sheer1, Jun Wei4, Anantha Marisetty4, Amy B Heimberger4, Robert L Bowman5, Sebastian Brandner2, Johanna A Joyce6 and Silvia Marino *,1 1Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, UK 2National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK 3Division of Neuropathology, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK 4Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA 6Department of Oncology, Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland *Corresponding author. Tel:+44 2078 822585; E-mail: [email protected] EMBO J (2020)39:e103790https://doi.org/10.15252/embj.2019103790 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Tumour-associated microglia/macrophages (TAM) are the most numerous non-neoplastic populations in the tumour microenvironment in glioblastoma multiforme (GBM), the most common malignant brain tumour in adulthood. The mTOR pathway, an important regulator of cell survival/proliferation, is upregulated in GBM, but little is known about the potential role of this pathway in TAM. Here, we show that GBM-initiating cells induce mTOR signalling in the microglia but not bone marrow-derived macrophages in both in vitro and in vivo GBM mouse models. mTOR-dependent regulation of STAT3 and NF-κB activity promotes an immunosuppressive microglial phenotype. This hinders effector T-cell infiltration, proliferation and immune reactivity, thereby contributing to tumour immune evasion and promoting tumour growth in mouse models. The translational value of our results is demonstrated in whole transcriptome datasets of human GBM and in a novel in vitro model, whereby expanded-potential stem cells (EPSC)-derived microglia-like cells are conditioned by syngeneic patient-derived GBM-initiating cells. These results raise the possibility that microglia could be the primary target of mTOR inhibition, rather than the intrinsic tumour cells in GBM. Synopsis Using glioblastoma multiforme (GBM) mouse models and human in vitro assays, this study identifies the mTOR pathway in microglia as a major regulator of immune evasion in the tumour stroma, pointing to a need for cell-targeted therapeutic approaches in brain malignancies. GBM patient-conditioned medium increases mTOR signalling in microglia but not bone-marrow-derived macrophages. Genetic mTORC1 inactivation in microglia reduces tumour growth in vivo. Microglial mTORC1 promotes STAT3-mediated secretion of anti-inflammatory cytokines and limits peripheral T cell infiltration. Syngeneic GBM-conditioned media deregulates mTOR signaling in human PSC-derived microglial-like cells. Introduction No effective therapy currently exists for glioblastoma (GBM), which is the most common primary brain tumour and one of the most aggressive types of cancers. Challenges in tackling these tumours are manifold, including their inter- and intratumour heterogeneity, the limited accessibility of systemically administered drugs, their infiltrative growth pattern and the complexity of the microenvironment in which they are embedded (Aldape et al, 2019). The contribution of the tumour microenvironment (TME), which is shaped by the communication between tumour cells and non-malignant cells, is undisputed in GBM pathogenesis (Quail & Joyce, 2017). Particular emphasis has been placed on immune infiltrates, including tumour-associated microglia/macrophages (TAM), which are the most numerous infiltrating cell population in GBM (Szulzewsky et al, 2015; Chen et al, 2017; Darmanis et al, 2017; Roesch et al, 2018). These cells engage in a bidirectional interaction with tumour cells to promote several aspects of glioma development, including proliferation, angiogenesis, immune evasion and therapeutic resistance (Hambardzumyan et al, 2016; Chen & Hambardzumyan, 2018). TAM in GBM are pro-tumourigenic, with increased accumulation in high-grade gliomas that correlates with poor prognosis (Komohara et al, 2008; Hambardzumyan et al, 2016; Sorensen et al, 2018). Moreover, TAM produce low levels of pro-inflammatory cytokines and lack key molecular mechanisms necessary for T-cell stimulation, suggesting a suppression of T-cell activation capacity in GBM (Hussain et al, 2007; Quail & Joyce, 2017). TAM can be classified into tumour-associated microglia (TAM-MG), endogenous to central nervous system (CNS) tissue and tumour-associated macrophages (TAM-BMDM), originating from bone marrow-derived monocytes that infiltrate the tumour from the periphery (Muller et al, 2015; Bowman et al, 2016; Haage et al, 2019). The functional contribution of TAM to GBM pathogenesis is well documented; however, it is unclear how each of these two ontogenetically distinct populations differentially contribute to the GBM phenotype. Efforts are being invested in therapeutically depleting immune cells from the TME as well as altering cytotoxic potential with immunomodulation (Seoane, 2016). Targeting chemokines and their receptors such as the CCR2/CCL2 axes has been explored as a therapeutic strategy to inhibit infiltration of TAM (Ruffell & Coussens, 2015; Vakilian et al, 2017). For the re-education of TAM immune activity, inhibition of colony-stimulating factor 1 receptor (CSF1R) has shown promising result in preclinical GBM models by blocking tumour growth and progression (Pyonteck et al, 2013; Yan et al, 2017). However, acquired resistance and tumour relapse emerge following long-term exposure to these therapies (Quail et al, 2016). To design successful re-education strategies targeting TAM, a better characterisation of their signalling mechanisms is essential. The mTOR pathway has been extensively studied in the context of cell growth, proliferation and survival in many cancers, including GBM (Li et al, 2016; Jhanwar-Uniyal et al, 2019). The central component of the pathway, the mTOR protein kinase, forms the catalytic subunit of the protein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which regulate different branches of the mTOR network (Shimobayashi & Hall, 2014; Yuan & Guan, 2016). mTORC1 signalling integrates inputs from inflammatory and growth factors as well as amino acids, energy status, oxygen levels and cellular stress pathways. The two major substrates of mTORC1 are p70 ribosomal protein S6 Kinase 1 (p70S6K1) and the eukaryotic translation initiation factor (eIF)-binding protein 1 (4EBP1; LoRusso, 2016). These signalling molecules impact on cell growth and metabolism, in part by increasing the biosynthesis of the cellular translational apparatus (Thoreen et al, 2012). The small GTPase Ras homolog enriched in brain (Rheb) is the only known direct activator of mTORC1. Conversely, the signalling pathways that lead to mTORC2 activation are not characterised in such detail. mTORC2 is known to regulate cell cycle entry, cell survival and actin cytoskeleton polarisation through its most common downstream substrates: AKT, SGK and PKC (Yang et al, 2013). Despite their biochemical and functional differences, crosstalk has been reported between the two complexes, which contributes to the modulation of their activity (Xie & Proud, 2014). In GBM, altered mTORC1 signalling activity correlates with increased tumour grade and is associated with poor prognosis (Duzgun et al, 2016). Consequently, mTOR kinase inhibitors targeting both mTORC1 and mTORC2 are considered promising anti-cancer therapies and are being tested in clinical trials, in combination with radiation and chemotherapy (Zhao et al, 2017; Mecca et al, 2018). In the last decade, extensive work has been carried out to characterise mTOR-dependent signalling in innate immune cells and its role in regulating the expression of inflammatory factors, antigen presentation, phagocytic activity, cell migration and proliferation (Weichhart et al, 2008; Jones and Pearce, 2017). mTOR signalling is known to regulate the balance between pro- and anti-inflammatory responses and may be responsible for the dysregulated inflammatory response in TAM, which display a shift towards anti-inflammatory activity. Interestingly, increased mTOR phosphorylation at Ser-2448 is present in nearly 40% of TAM in human GBM (Lisi et al, 2019); however, the functional impact of this mTOR deregulation and its molecular mechanism have never been characterised. Here, we have used GBM orthotopic allografts in genetically engineered mice in which mTORC1 signalling has been silenced in TAM-MG, as well as human expanded-potential stem cells (EPSC)-derived microglial-like cells and matched GBM cells to study the role of the mTOR pathway in TAM-MG in the GBM microenvironment. Results mTOR signalling is upregulated in TAM-MG but not TAM-BMDM in GBM mouse models To determine whether mTOR signalling was deregulated in TAM, we assessed the activity of the pathway in GBM mouse models that recapitulate the genetic signatures of human GBM: GL261 allograft model, Ntv-a;PDGFB + Shp53 (Bowman et al, 2016), Pten−/−; p53−/− (Jacques et al, 2010), Pten−/−; p53−/−; Idh1R132H and PDGFB genetic model (Zhang et al, 2019). Tumours were stained for ionised calcium binding adaptor molecule 1 (Iba1, a marker of TAM) and for phosphorylated S6 (p-S6; S240/244, a downstream marker of mTORC1 activity). Co-expression of these markers, as defined by the fraction of Iba1+ cells expressing p-S6, was quantified. Three regions—non-tumour brain tissue, tumour edge and tumour core—were defined and analysed separately, to account for potentially different functional properties of TAM within the tumour as compared to the surrounding brain, as previously reported (Darmanis et al, 2017; Fig 1A). Tumour core regions were defined as highly cellular areas composed almost entirely of tumour cells (cells with marked pleomorphism and nuclear atypia—increased nuclear size and hyperchromasia—as well as mitotic activity on the haematoxylin and eosin (H&E) staining). The tumour edge refers to the infiltration zone, while areas without tumour infiltration were defined as non-tumour brain tissue (Fig 1A). As expected, Iba1+ cells showed a ramified morphology with long, thin cellular processes and small cell bodies in non-tumour areas, while they displayed shorter and fewer processes, with rounder cell bodies at the tumour edge. In the tumour core, they acquired an amoeboid shape, without branched processes and large round cell bodies (Fig 1B). Quantification showed that p-S6+ Iba1+ cells were extremely low in the non-tumour tissue (ranging from 1% (± 0.2 SEM) in the GL261 model to 13.9% (± 7.4 SEM) in the PDGFB model), whereas co-expression was more frequent in the tumour core (between 50% and 83%, with the PDGFB model at 83% (± 9.9 SEM) closely followed by the GL261 model at 71% (± 8 SEM); Fig 1B). In the tumour edge regions, all models except Ntv-aPDGFB + Shp53 showed significant difference in the fraction of Iba1+ cells co-expressing p-S6 as compared to non-tumour tissue (between 31% (± 4 SEM) the GL261 model and 72% (± 14 SEM) in the Pten−/−; p53−/− model; Fig 1B). We conclude that increased mTOR activity was consistently observed in TAM across tumour models, and it was predominantly independent of intratumour location. Figure 1. Upregulation of mTOR signalling in TAM-MG but not TAM-BMDM in GBM mouse models A. Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue). B. Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten−/−p53−/− (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high-grade glioma models were analysed—GL261 (n = 3), PDGFB (n = 3), Ntv-a;PDGFB+shp53 (n = 2), Pten−/−p53−/− (n = 3) and Pten−/−p53−/−Idh1mut (n = 3) (mean ± SEM; two-way ANOVA Tukey test). C. Venn diagram identifying significantly deregulated genes in healthy versus GL261 TAM-MG and/or TAM-BMDM (GSE68376 dataset). D. Top-most deregulated canonical pathways in GL261 TAM-MG, as identified by the IPA software. Threshold indicates P ≤ 0.05. Z-score indicates the orientation of the deregulation. Ratio indicates the number of deregulated genes in the pathway. E. mTOR-related deregulated canonical pathways in GL261 TAM-MG and TAM-BMDM, as identified by the IPA software. F, G. Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n = 3; mean ± SEM, likelihood ratio test in edgeR). Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Download figure Download PowerPoint Taking advantage of transcriptomic data from TAM-MG and TAM-BMDM isolated from GL261 tumours (Bowman et al, 2016), deregulation of the mTOR pathway was further investigated in these two cell populations. 4,907 and 5,089 deregulated genes were identified in the comparison between healthy microglia and TAM-MG, and between blood monocytes and TAM-BMDM, respectively. Amongst these deregulated genes, the majority were unique to TAM-MG and TAM-BMDM (3,405 and 3,687, respectively), in keeping with the different ontogeny of these cells (Fig 1C). The most significantly deregulated pathways identified by ingenuity pathway analysis (IPA; Kramer et al, 2014) in TAM-MG included mTOR signalling and mTOR-related signalling pathways: EIF2 signalling, regulation of eIF4 and p70S6K signalling, and mTOR signalling (Fig 1D). These pathways, as indicated by positive Z-score, were predicted to be activated (Fig 1D, Appendix Fig S1A and Dataset EV1). Importantly, these pathways were not detected as enriched when looking at the 5,189 deregulated genes in TAM-BMDM (Fig 1E, Appendix Fig S1B), and upregulation of RPS6 and EI4EBP1 was detected in TAM-MG but not in TAM-BMDM compared to controls (Fig 1F and G). These results show an increase in mTOR activity in TAM in several mouse models of GBM, which is specific to TAM-MG and not observed in TAM-BMDM in the GL261 model. Glioblastoma initiating cells increase mTOR signalling via PI3K/AKT axis in tumour-conditioned microglia but not BMDM Next, we asked how mTOR deregulation occurred in TAM-MG and whether glioblastoma initiating cells (GIC) could play a role, considering that they secrete growth and inflammatory factors that could potentially stimulate mTOR signalling in microglia (Okawa et al, 2017). We tested this hypothesis in an in vitro setting, where primary microglia and bone marrow-derived macrophages (BMDM), harvested from neonatal and 3-month-old C57BL/6 mice, were conditioned with the supernatant from different primary patient-derived GIC lines. Conditioned media was obtained from GL261 (GL261-CM) and primary Pten−/−;p53−/− mGIC cultures (mGICPten−/−;p53−/−-CM), two models with increased mTOR signalling in TAM-MG in vivo (Fig 1B–G). The secretome of mouse neural stem cells (mNSC-CM) derived from syngeneic mice was used as a control (Fig 2A). Unconditioned microglia and BMDM cultures were also used as controls (Fig 2A). Figure 2. Microglia and BMDM are differently conditioned by mGIC A. Schematic of the in vitro model whereby microglia and BMDM were pretreated with Torin, LY294002 as indicated and stimulated with mGL261, mGICPten−/−;p53−/−-CM or mNSC-CM. B–I. Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4 h (B, D and F) and 0.5 h (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244; (E) p-4EBP1 T37/46; (G) p-AKT S473; and (I) p-AKT T308. Each treatment (mNSC-CM, Pten−/−p53−/− mGIC-CM, Pten−/−p53−/− mGIC-CM+Torin, mGIC-CM+LY) was normalised to unconditioned control (n = 3; mean ± SEM, two-way ANOVA Tukey test). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 comparing mGICPten−/−;p53−/−-CM versus mGICPten−/−;p53−/−-CM +inhibitor. Source data are available online for this figure. Source Data for Figure 2 [embj2019103790-sup-0009-SDataFig2.zip] Download figure Download PowerPoint mTORC1 specifically phosphorylates S6 at S240/244 and 4EBP1 at T37/46. Microglia conditioned with mGICPten−/−;p53−/−-CM or GL261-CM showed a significant increase in phosphorylation at both of these sites (Figs 2B and D, and EV1A), when compared to cultures treated with mNSC-CM or unconditioned media, supporting the notion that the phenotype is consistent across glioblastoma models. Conversely, BMDM displayed no difference in p-S6 or p-4EBP1 levels in cultures treated with mGICPten−/−;p53−/−-CM or GL261-CM, compared to mNSC-CM or unconditioned media (Fig EV1C and E). Flow cytometry analysis of these cultures confirmed the increase in p-S6 (Figs 2C and EV1B) and p-4EBP1 (Figs 2E and EV1B) levels in mGICPten−/−;p53−/−-CM-treated microglia but not BMDM (Fig EV1F) compared to the mNSC-CM treatment (normalised to unconditioned cultures). Increased phosphorylation of the upstream regulator AKT at S473 sites in microglia (Figs 2F and EV1A) but not BMDM (Fig EV1C and E) treated with mGICPten−/−;p53−/−-CM or GL261-CM demonstrated increased activity of mTORC2 signalling. Treatment with Torin (inhibitor of mTORC1 and mTORC2) resulted in significant reduction p-AKT (S473) in tumour-conditioned microglia (Figs 2F and EV1A), a finding which was validated by FACS (Figs 2G and EV1B and F). Increased phosphorylation of AKT at T308 site was also detected (Fig 2H). Treatment with the PI3K inhibitor (LY294002) resulted in significant reduction in p-AKT (T308) and p-S6 (S240/244) in mGICPten−/−;p53−/−-CM and GL261-conditioned microglia (Figs 2H and I, and EV1A and B) but not in BMDM (Fig EV1D–F), thereby confirming activation of mTOR signalling via PI3K/AKT in tumour-conditioned microglia. Click here to expand this figure. Figure EV1. Microglia and BMDM are differently conditioned by mGIC-secreted factors in vitro A. Quantification of immunoblotting analysis from conditioned microglia whole cell lysates at 4 h incubation for p-S6, p-4EBP1 and p-AKT S473 and at 0.5 h incubation for p-AKT T308. Data normalised to non-phosphorylated protein and to vinculin. B. Representative flow cytometry plots of microglia under different culture condition at 4 h incubation for p-S6, p-4EBP1 and p-AKT S473 and at 0.5 h incubation for p-AKT T308. C–E. Signalling was analysed in conditioned BMDM by immunoblotting of whole cell lysate collected at 4 h for p-S6, p-4EBP1 and p-AKT S473 (C) and at 0.5 h incubation for p-AKT T308 (D), which was quantified by normalisation with non-phosphorylated protein and vinculin (E). F. Flow cytometry analysis was carried out in BMDM for p-S6, p-4EBP1, p-AKT S473 and p-AKT T308. Each treatment was normalised to unconditioned control (n = 3; mean ± SEM; two-way ANOVA Tukey test). Source data are available online for this figure. Download figure Download PowerPoint These results show that factors secreted by mGICPten−/−;p53−/− and GL261 cultures upregulate mTORC1 and mTORC2 signalling via the PI3K/AKT axes in microglia but not in BMDM. Genetic inhibition of mTORC1 signalling in Cx3cr1+ TAM reduces tumour growth and increases survival To investigate the functional role of activated mTOR signalling in TAM-MG, genetic inhibition was established in these cells in mice recipient of GL261 GBM allografts (Fig 3A). The GL261 cell line was chosen to generate fast-growing orthotopic syngeneic GBM models in immunocompetent mice. A mouse line with a floxed exon 3 of the Rheb1 gene (Rheb1fl/fl), a key effector of mTOR, was chosen to inactivate the pathway in TAM. Genetic modulation of mTOR signalling in vivo was achieved by crossing the Rheb1fl/fl mice with Cx3cr1-CreERT2 knock-in mice, resulting in deletion of Rheb1 in microglia upon tamoxifen-induced Cre expression. Three weeks after tamoxifen injection, GL261 tumour cells were injected intracerebrally in mutant animals as well as in controls lacking the Cre construct but which also had received tamoxifen treatment (Fig 3A). Mice were culled when symptomatic and a longer survival was observed for the Cx3cr1- Rheb1Δ/Δ mice as compared to the Rheb1fl/fl mice (Fig 3B). An independent cohort of allografted mice was generated and imaged 20 days post-tumour initiation by MRI. In this cohort, measurement of tumour volume confirmed that tumours were significantly smaller in the Cx3cr1-Rheb1Δ/Δ compared to Rheb1fl/fl mice (Fig 3C). The experiment was terminated 25 days post-tumour initiation and the brains either processed for histology (n = 5) or analysed by flow cytometry (n = 6). Histological features were those of a highly cellular glial tumour with prominent nuclear pleomorphism, brisk mitotic activity and multifocal microvascular proliferations (Fig 3D). While no histological differences were noted between the two genotypes (Fig 3D), the numbers of GL261 GFP+ tumour cells were lower in the Cx3cr1-Rheb1Δ/Δ tumours (Fig 3E), consistent with the reduced tumour volume observed by MRI. Figure 3. Genetic inhibition of mTORC1 signalling in Cx3cr1+ TAM impacts tumour growth and survival Schematic of the generation and analysis of the Cx3cr1-Cre;Rheb1-loxp GL261 model. Survival analysis for Cx3cr1-Rheb1Δ/Δ (n = 7) and Rheb1fl/fl (n = 7) mice. Chi-square test. Representative images and quantification of the tumour volume with MRI of Rheb1fl/fl (n = 8) compared to Cx3cr1-Rheb1Δ/Δ (n = 8) mice (mean ± SEM; unpaired parametric t-test). H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images) Percentage of GFP+ CD45− GL261 tumour cells in Cx3cr1-Rheb1Δ/Δ (n = 5) and Rheb1fl/fl (n = 7) tumours, with representative FACS plot (mean ± SEM; unpaired parametric t-test). Data information: **P ≤ 0.01, and ***P ≤ 0.001. Download figure Download PowerPoint Overall 98% of TAM-MG were positive for YFP, the expression of which was dependent on Cre expression in Cx3cr1-Rheb1Δ/Δ mice (Fig EV2A–C). Additionally, 35% of TAM-BMDM expressed YFP (Fig EV2A–C), in accordance with the known expression pattern of the Cx3cr1 promoter in these tumours (Bowman et al, 2016). mTOR inhibition was confirmed in the tumours by assessing the expression levels of p-S6 in P2RY12+ CD49d− TAM-MG and P2RY12− CD49d+ TAM-BMDM by flow cytometry. We observed that p-S6 baseline levels in Rheb1fl/fl tumours were higher in TAM-MG than TAM-BMDM, and these were significantly reduced in TAM-MG but not in TAM-BMDM in Cx3cr1-Rheb1Δ/Δ tumours (Fig EV2A–C). A clear reduction in the number of Iba1+ p-S6+ cells was also seen in the Cx3cr1-Rheb1Δ/Δ versus Rheb1fl/fl tumour tissues (Fig EV2D and E). Click here to expand this figure. Figure EV2. Cx3cr1-Rheb1Δ/Δ mice display inhibition of mTORC1 signalling in GL261 TAM Representative flow cytometry plot for the expression of CD45 and CD11b in GL261 tumour, with CD45high CD11b+ TAM-BMDM (top gate) and CD45low CD11b+ TAM-MG (lower gate). Representative flow cytometry plot of YFP (left) and p-S6 (right) levels in TAM-BMDM (top) and TAM-MG (bottom) in Cx3cr1-Rheb1Δ/Δ (blue) versus Rheb1fl/fl (red) GL261 tumours. Percentage of TAM-MG and TAM-BMDM expressing YFP in Cx3cr1-Rheb1Δ/Δ (n