Glioblastoma hijacks neuronal mechanisms for brain invasion.

Glioblastomas are incurable tumors infiltrating the brain. A subpopulation of glioblastoma cells forms a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs). Other subpopulations appear unconnected, and their biological role remains unclear. Here, we demonstrate that whole-brain colonization is fueled by glioblastoma cells that lack connections with other tumor cells and astrocytes yet receive synaptic input from neurons. This subpopulation corresponds to neuronal and neural-progenitor-like tumor cell states, as defined by single-cell transcriptomics, both in mouse models and in the human disease. Tumor cell invasion resembled neuronal migration mechanisms and adopted a Lévy-like movement pattern of probing the environment. Neuronal activity induced complex calcium signals in glioblastoma cells followed by the de novo formation of TMs and increased invasion speed. Collectively, superimposing molecular and functional single-cell data revealed that neuronal mechanisms govern glioblastoma cell invasion on multiple levels. This explains how glioblastoma's dissemination and cellular heterogeneity are closely interlinked.

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.

Advancing preclinical cancer models to assess clinically relevant outcomes.

Cancer models are indispensable research tools for elucidating the mechanisms involved in tumor onset, progression and treatment resistance. They are key in evaluating therapeutics prior clinical trials. In this editorial, we invite contributions for a BMC Cancer's Collection of articles addressing 'Advances in pre-clinical cancer models' towards relivable outcomes at the preclinical stage.

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

Complete polyp resection with cold snare versus hot snare polypectomy for polyps of 4-9 mm: a randomized controlled trial.

BACKGROUND: Endoscopic screening with polypectomy reduces the incidence of colorectal cancer (CRC). Incomplete polyp removal may attenuate the effect of screening. This randomized trial compared cold snare polypectomy (CSP) with hot snare polypectomy (HSP) in terms of complete polyp resection. METHODS: We included patients ≥ 40 years of age at eight hospitals in four countries who had at least one non-pedunculated polyp of 4-9 mm detected at colonoscopy. Patients were randomized 1:1 to CSP or HSP. Biopsies from the resection margins were obtained systematically after polypectomy in both groups. We hypothesized that CSP would be non-inferior to HSP, with a non-inferiority margin of 5 %. Logistic regression models were fitted to identify the factors explaining incomplete resection. RESULTS: 425 patients, with 601 polyps, randomized to either CSP or HSP were included in the analysis. Of 318 polyps removed by CSP and 283 polyps removed by HSP, 34 (10.7 %) and 21 (7.4 %) were incompletely resected, respectively, with an adjusted risk difference of 3.2 % (95 %CI -1.4 % to 7.8 %). There was no difference between the groups in terms of post-polypectomy bleeding, perforation, or abdominal pain. Independent risk factors for incomplete removal were serrated histology (odds ratio [OR] 3.96; 95 %CI 1.63 to 9.66) and hyperplastic histology (OR 2.52; 95 %CI 1.30 to 4.86) in adjusted analyses. CONCLUSION: In this randomized trial, non-inferiority for CSP could not be demonstrated. Polyps with serrated histology are more prone to incomplete resection compared with adenomas. CSP can be used safely for small polyps in routine colonoscopy practice.

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.

Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures.

Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglial responses are thought to worsen CNS diseases; nevertheless, their impact during neuroinflammatory processes remains largely obscure. Here, using a combination of single-cell RNA sequencing and multicolour flow cytometry, we comprehensively profile microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we show that microglia isolated from LPS-injected mice display a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identify distinct microglial activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglial heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases.

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples.

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.

Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas.

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes.

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.

Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.

The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the 'side population' phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.

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.

Immune cell identity behind the Ktrans mapping of mouse glioblastoma.

Dynamic contrast-enhanced MR imaging (DCE-MRI) can assess the integrity of the blood brain barrier (BBB) and has been used in GBM patients to determine glioma grade, predict prognosis, evaluate treatment response, and differentiate treatment-induced effect from recurrence. The volume transfer constant Ktrans is the most frequently used metric in tumor assessment. Based on previous studies that a higher WHO grade of brain tumor was associated with greater impairments of immunity and that Ktrans value was associated with the pathological grading, the relationship between differential composition of immune cells in GBM tissue and dynamic changes in Ktrans mapping was anticipated in this study. The present study utilized an orthotopic allograft model of GBM in which mouse GL26 cells are implanted into Ccr2RFP/wtCx3cr1GFP/wt mice on a C57 background. The brain tumors exhibited heterogenous Ktrans values with the coefficients of variation (CV) above 75%, or relatively homogeneous Ktrans maps with CV values below 50%. The Ktrans values of homogeneous tumors ranged between 0.02/min-0.32/min with a median value of 0.10/min. The immune cell composition defined by quantitative immunohistochemistry and cell sorting was compared between the tumors with Ktrans values above 0.10/min (higher Ktrans) or below 0.10/min (lower Ktrans). Histological analysis showed that tumors with higher Ktrans values exhibited greater numbers of CCR2pos cells (257.60 ± 16.42/mm2 vs 203.23 ± 12.20/mm2, p = 0.04) and an increased ratio of CCR2pos cells to CX3CR1pos cells (1.20 ± 0.02 vs 0.38 ± 0.04, p = 0.001), the numbers of CX3CR1pos cells did not differ significantly based on Ktrans values (219.70 ± 16.20/mm2 vs 250.38 ± 21.20/mm2, p = 0.19). Flowcytometry analysis showed that tumors with higher Ktrans values (above 0.1/min) were associated with greater numbers of both overall monocytes (54.93 ± 6.81% vs 29.75 ± 3.54%, p = 0.01) and inflammatory monocytes (72.38 ± 1.49% vs 59.52 ± 2.44%, p = 0.001). In contrast, tumors with lower Ktrans values (below 0.1/min) exhibited greater numbers of patrolling monocytes (75.65 ± 4.14% vs 63 ± 6.94%, p = 0.05). In the tumors with lower Ktrans values, all three types of tumor associated cells, including patrolling monocytes, inflammatory monocytes, and microglia cells possessed a higher proportion of cells at pro-inflammatory status (41.77 ± 6.13% vs 25.06 ± 6.72%, p = 0.05; 27.50 ± 2.11% vs 20.62 ± 1.87%, p = 0.03; and 55.80 ± 9.88% vs 31.12 ± 7.31%, p = 0.05), inflammatory monocytes showed fewer anti-inflammatory cells (1.25 ± 0.62% vs 3.16 ± 3.56%, p = 0.04). Taken together, differences in Ktrans values were associated with differential immune cell phenotypes and polarizations. Ktrans mapping may therefore represent a novel approach for defining the immune status of GBM.

Molecular Identity Changes of Tumor-Associated Macrophages and Microglia After Magnetic Resonance Imaging-Guided Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Mouse Glioblastoma Model.

An orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood-brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS had significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells. The effect of MRgFUS on immune cell composition was also characterized and quantified using flow cytometry. Glioma implantation resulted in increased amounts of lymphocytes, monocytes and neutrophils in the brain parenchyma. Tumors administered MRgFUS exhibited increased numbers of monocytes and monocyte-derived TAMs. In addition, MRgFUS-treated tumors exhibited more CD80+ cells in monocytes and microglia. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes and induce a shift toward a more pro-inflammatory status of the immune environment in glioblastoma.

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.

Cancer cell heterogeneity and plasticity: A paradigm shift in glioblastoma.

Phenotypic plasticity has emerged as a major contributor to intra-tumoral heterogeneity and treatment resistance in cancer. Increasing evidence shows that glioblastoma (GBM) cells display prominent intrinsic plasticity and reversibly adapt to dynamic microenvironmental conditions. Limited genetic evolution at recurrence further suggests that resistance mechanisms also largely operate at the phenotypic level. Here we review recent literature underpinning the role of GBM plasticity in creating gradients of heterogeneous cells including those that carry cancer stem cell (CSC) properties. A historical perspective from the hierarchical to the nonhierarchical concept of CSCs towards the recent appreciation of GBM plasticity is provided. Cellular states interact dynamically with each other and with the surrounding brain to shape a flexible tumor ecosystem, which enables swift adaptation to external pressure including treatment. We present the key components regulating intra-tumoral phenotypic heterogeneity and the equilibrium of phenotypic states, including genetic, epigenetic, and microenvironmental factors. We further discuss plasticity in the context of intrinsic tumor resistance, where a variable balance between preexisting resistant cells and adaptive persisters leads to reversible adaptation upon treatment. Innovative efforts targeting regulators of plasticity and mechanisms of state transitions towards treatment-resistant states are needed to restrict the adaptive capacities of GBM.

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.

Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration.

Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.