bluuebunny/crossref_metadata_2025 huggingface dataset:
TAMI-09. INTRAOPERATIVE MICRODIALYSIS AS A FEASIBLE PLATFORM FOR METABOLIC AND PHARMACODYNAMIC BIOMARKER DISCOVERY
Authors: Burns Terence , Riviere-cazaux Cecile , Shoushtarizadeh Alireza (Ali) , Rajani Karishma , Rahman Masum , Ian Olson , Carlstrom Lucas
Published: November 2021

BACKGROUND

Progress for gliomas is slowed in part by the paucity of mechanistic feedback during treatment with experimental therapies. Access to extracellular tumor pharmacodynamic biomarkers could provide an avenue to accelerate progress. We have initiated a program of intra-operative microdialysis to accelerate biomarker discovery and to identify candidate outcome measures for translational therapies.

METHODS

Intraoperative microdialysis was performed with M-dialysis 100kDA catheters and 107 variable rate pumps under an IDE. Four IDH-mutant and two IDH-WT lesions were studied intraoperatively with 3 divergently placed catheters. Microperfusate (artifical CSF+ 3% dextran) was perfused at 2uL/min and collected in 20 min increments. Paired CSF was also obtained when accessible. A parallel cohort of nude mice bearing human IDH-mutant, IDH-WT, or sham intracranial xenografts (n=6-12) underwent intratumoral microdialysis. A pilot murine study of intracranial drug delivery was performed via concurrent microdialysis during convection-enhanced delivery (CED) of saline or the IDH-inhibitor AG120.

RESULTS

Microdialysate from IDH-mutant intracranial xenografts revealed >100 differentially abundant metabolites compared to sham or IDH-WT tumors, including D2-HG (21x) and MTA(18x), p< 10^-5. The most significantly abundant metabolite was DMA (4x, p< 10^-10). 15-1000uM D2HG was recovered from intra-operative human IDH-mutant tumors and 1-2uM from normal brain adjacent to IDH-WT gliomas and < 1uM in all IDH-WT samples. Forty metabolites differentiated enhancing tumor from adjacent brain in 3/3 paired human samples including upregulated Aminoacyl-tRNA biosynthesis and downregulated purine metabolism. Serial aliquots of microdialysate during saline CED yielded steady D2-HG levels whereas CED with AG120 yielded undetectable D2-HG within 6 hours.

CONCLUSION

The extracellular metabolic landscape of glioma is diverse, dynamic and reflects tumor biology and response to therapy. Collectively, these studies suggest that intra-tumoral drug testing should be feasible with realistic expectation of gaining metabolic feedback within a short timeframe. Leveraging this paradigm can provide opportunities to accelerate therapeutic translation for gliomas.
TAMI-12. CANCER-IMMUNE CELL INTERACTIONS DRIVE TRANSITIONS TO MESENCHYMAL-LIKE STATES IN GLIOBLASTOMA
Authors: Hara Toshiro , Chanoch-Myers Rony , Mathewson Nathan , Myskiw Chad , Atta Lyla , Bussema Lillian , Eichhorn Stephen , Greenwald Alissa , Kinker Gabriela , Rodman Christopher , Castro L Nicolas Gonzalez , Wakimoto Hiroaki , Rozenblatt-Rosen Orit , Zhuang Xiaowei , Fan Jean , Hunter Tony , Verma Inder , Wucherpfennig Kai , Regev Aviv , Suva Mario , Tirosh Itay
Published: November 2021

Communication between cancer cells and immune cells is a key determinant of the glioblastoma ecosystem and its response to therapies, but remains poorly understood. Here we leveraged single-cell RNA-sequencing (scRNA-seq) of human samples and mouse models, deconvolution analysis of bulk specimens from The Cancer Genome Atlas (TCGA) and functional approaches to dissect cellular states and cross-talk in glioblastoma. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived Oncostatin M (OSM) and its cognate receptor OSMR on glioblastoma cells. We show that MES-like glioblastoma states are associated with increased T cells cytotoxicity and potentially with better clinical response to immunotherapies. Overall, our work dissects the cellular interactions within the glioblastoma microenvironment, with potential implications for therapies.
TAMI-26. INVESTIGATING TUMOR MICROENVIRONMENT METABOLIC DEPENDENCIES IN GLIOBLASTOMA
Authors: Minami Jenna , Bayley Nicholas , Tse Christopher , Morrow Danielle , Zhu Henan , Yong William , Liau Linda , Cloughesy Timothy , Graeber Thomas , Nathanson David
Published: November 2021

Metabolic reprogramming is a hallmark of cancer. Malignant cells must acquire metabolic adaptations in response to a multitude of intrinsic and extrinsic factors to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Altered metabolism in GBM is becoming an increasingly promising area of research to identify novel therapeutic targets and biomarkers, as metabolic rewiring can occur across numerous genotypes. The unique features of the brain TME pose a difficult challenge when studying GBM and other primary brain cancers – currently, the availability of nutrients in the brain, as well as how they influence or are influenced by tumor metabolism, are not well understood. Our group has identified a subgroup of gliomas, hereafter termed TME-dependent, which can only form tumors in the brain TME. While genetically heterogeneous, these tumors share transcriptional identities linked to oligodendrocyte precursor cell (OPC) and neuronal lineages. Systematic molecular profiling of over 75 patient tumors and their matched cell culture and brain orthotopic xenograft derived models revealed that TME-dependent tumors display lipid metabolic signatures linked to signaling and interactions with surrounding neurons and glial cells. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity in fatty acid biosynthetic programs, indicating a potential brain-microenvironment specific metabolic dependency linked to transcriptional identity that can be targeted for therapy.
TAMI-29. MR SPECTROSCOPY MEASURES OF LAC/NAA AND NAA/CHO DIFFERENTIATE SURVIVORSHIP IN PATIENTS WITH RECURRENT GLIOBLASTOMA TREATED WITH ANTI-ANGIOGENIC THERAPY
Authors: Talati Pratik , El-Abtah Mohamed , Kim Daniel , Dietrich Jorg , Fu Melanie , Wenke Michael , He Julian , Natheir Sharif , Vangel Mark , Rapalino Otto , Vaynrub Anna , Arrillaga-Romany Isabel , Forst Deborah , Yen Yi-Fen , Andronesi Ovidiu , Kalpathy-Cramer Jayashree , Rosen Bruce , Batchelor Tracy , Gonzalez Ramon , Gerstner Elizabeth , Ratai Eva-Maria
Published: November 2021

Patients with recurrent glioblastoma (rGBM) are often started on anti-angiogenic therapy such as bevacizumab. However, determining treatment failure using conventional MRI methods remains challenging. We prospectively collected longitudinal MR spectroscopy data in 33 patients with rGBM and quantified various metabolites including N-acetylaspartate (NAA), Choline (Cho), and Lactate (Lac). After stratifying patients by 9 month survival, we found that longer-term survivors had decreased Lac/NAA and increased NAA/Cho compared to shorter-term survivors. ROC analyses illustrated that intratumoral changes in NAA/Cho were predictive of survival at 1 day (AUC 0.92), 2 weeks (AUC 0.75), 8 weeks (AUC 0.71) and 16 weeks (AUC 0.85) but not 4 weeks (AUC 0.60). Intratumoral changes in Lac/NAA were predictive of survival at all time points tested (AUCs > 0.76 for all time points). At 8 weeks, 90% of patients with increased Lac/NAA from baseline and 88% of patients with decreased NAA/Cho did not survive 9 months. Changes in NAA/Cho and Lac/NAA may serve as early biomarkers of anti-angiogenic treatment failure.
TAMI-34. TARGETING CSF1R AND PD1 IN EXPERIMENTAL GLIOMA
Authors: Becker Hannes , Przystal Justyna M , Canjuga Denis , Tsiami Foteini , Anderle Nicole , Keller Anna-Lena , Pohl Anja , Ries Carola H , Schmittnaegel Martina , Korinetska Nataliya , Koch Marilin , Schittenhelm Jens , Tatagiba Marcos , Schmees Christian , Beck Susanne C , Tabatabai Ghazaleh
Published: November 2021

The treatment of glioblastoma remains a challenge. Novel therapeutic strategies are urgently needed. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting approach in this regard. Tumor-associated macrophages represent a distinct population of tumor-infiltrating immune cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival inside the tumor microenvironment. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We detected CSF1R expression in paired tissue samples of primary and corresponding progressive glioblastoma. We investigated anti-CSF1R treatment alone or in combination with anti-PD1 antibodies in the orthotopic syngeneic VM/Dk SMA560 glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies prolonged the latency until the onset of neurological symptoms and led to long-term survivors in vivo. Immunohistochemical analysis of post-treatment SMA-560 glioma revealed a modulation of the microenvironment, including increased T cell infiltration and reduced numbers of tumor-associated macrophages. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Taken together, our data indicates that CSF1R is a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.
TAMI-38. TUMOR-ASSOCIATED NEUTROPHILS IN GLIOBLASTOMA PROMOTE THE PERIVASCULAR GLIOMA STEM-LIKE CELL NICHE VIA OSTEOPONTIN SECRETION
Authors: Beniwal Angad , Jain Saket , Shah Sumedh , Gill Sabraj , Yagnik Garima , Nguyen Alan , Wadhwa Harsh , Diaz Aaron , Aghi Manish K
Published: November 2021

Among clinical analyses, elevated neutrophil-lymphocyte ratio has been correlated with poor outcomes of glioblastoma patients independent of other prognostic factors. Additionally, our flow cytometric studies of primary patient samples found neutrophil percentage to be significantly higher in higher-grade glioma versus lower-grade glioma. Tumor-associated neutrophils (TANs) comprise less than 2% of the glioblastoma microenvironment. While TANs were initially considered passive bystanders due to their short-lived nature, investigation of TANs in other cancers revealed distinct pro-tumoral roles. Therefore, we transcriptomically characterized glioblastoma TANs and defined their oncologic effects. Transcriptomic analysis of patient-matched TANs versus peripheral blood neutrophils revealed that functionally quiescent circulating neutrophils infiltrate IDH1-wild type glioblastoma via leukotriene B4 chemoattraction, where tumor cells morphologically and transcriptomically activate them to become TANs. Single-cell RNA-sequencing of patient-matched TANs and peripheral blood neutrophils revealed a subset of tumor-activated neutrophils which adopt a pro-tumoral secretory phenotype, marked by activation of the IL-17 signaling pathway and high osteopontin production. Using immunofluorescence stains of primary patient glioblastoma sections, we demonstrated that activated, myeloperoxidase-positive TANs reside in the perivascular niche of glioblastoma in close proximity to glioblastoma stem-like cells (GSCs) and CD31-positive endothelial cells. Further analysis in culture demonstrated that TAN-secreted osteopontin drives the formation, self-renewal, and proliferation of GSC-containing neurospheres. These results were validated using a syngeneic stem cell-derived IDH1-wild type murine glioblastoma model in vivo. Thus, while TANs are rare in glioblastoma, their enrichment in the glioblastoma perivascular niche uniquely positions them to support the GSCs that are crucial to therapeutic resistance of GBM.
TAMI-39. INTEGRATION OF SPATIALLY RESOLVED TRANSCRIPTOMICS AND METABOLOMICS UNCOVERS HYPOXIA-DRIVEN ACCUMULATION OF GENOMIC INSTABILITIES IN HUMAN GLIOMA
Authors: Kückelhaus Jan , Will Paulina , Ricklefs Franz , Benotmane Jasim Kada , Joseph Kevin , Beck Jürgen , Schnell Oliver , Ravi Vidyha , Heiland Dieter Henrik
Published: November 2021

High-dimensional technologies have provided insights into transcriptional heterogeneity and dynamic plasticity which are hallmarks of brain tumors. Although scRNA-seq recovers the diversity of transcriptional states, their spatial context within the neuronal environment has remained unexplored. Here, we integrated spatially resolved transcriptomics and metabolomics to characterize the glioma landscape at multiple molecular levels. We integrated spatial transcriptomics (10X Visium, n= 28) and metabolomics (MALDI, n= 6) from primary and recurrent glioblastoma patients. Unsupervised cluster analysis and pattern recognition uncovered 5 spatially distinct transcriptional programs, shared across patients. These included three cell-specific developmental stages largely reflecting those that are part of recently suggested models. By integrating metabolome data, we identified an additional program encompassing reactive responses to hypoxia. Areas of hypoxic response were negatively correlated with proliferation (R2= -0.34, p< 0.001) and significantly enriched for gene expression signatures from the S-phase (p< 0.001). Modeling of transient spatial gradients using vector field predictions showed opposing vector directions of hypoxia response and migratory capacity, underpinning the “go-or-growth” theory, where cells either proliferate or migrate. Inferred copy-number alterations (CNA) revealed a significant increase in genomic instability, highly correlated to hypoxia response (R2= 0.78, p< 0.001). Near necrotic areas, we observed a significant accumulation of CNAs while proliferation was inhibited, and cells remained in the S-phase. We validated this hypothesis of hypoxia-driven accumulation of CNAs by chronic hypoxia cultures of primary patient-derived cell lines. A gain of chromosomal instability after long-term hypoxia was observed, suggesting that hypoxic areas in glioblastoma function as bioreactors for genomic instability. Our findings elucidate the evolution of resistant subclones in glioblastoma. They provide novel insights into the dynamic regulation and interaction between host and tumor and cast a new light on hypoxic and necrotic areas, which may represent the source of the heterogeneous and resistant nature of glioblastomas.
TAMI-42. THE ROLE OF HFE AND IRON IN CELL ADHESION AND MIGRATION IN GLIOBLASTOMA
Authors: Shenoy Ganesh , Troike Katie M , Kuhn Madison , Webb Becky Slagle , Snyder Amanda , Khunsriraksakul Chachrit , Lathia Justin , Wang Hong-Gang , Proctor Elizabeth , Connor James
Published: November 2021

Glioblastoma (GBM) remains one of the most difficult to treat malignancies facing modern medicine. The strong migratory and invasive capacity of GBM cells allows for diffuse invasion into neighboring healthy brain which presents a significant hurdle for complete surgical resection of these tumors. Unsurprisingly, even after receiving maximal surgical resection, radiation and chemotherapy, the majority of GBM patients end up with recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in brain tumors such as GBM have been associated with poorer outcomes. In order to better understand how HFE expression impacts the adhesive and migratory capacity of GBM, we utilized syngeneic mouse glioma models (KR158, CT2A) that have been transfected to either over-express or under-express HFE. We observed that knocking down HFE in the KR158 model resulted in significantly decreased migratory capacity as well as decreased adhesion to fibronectin and artificial basement membrane. Likewise, overexpressing HFE in a CT2A model resulted in increased adhesion to fibronectin or artificial basement membrane. Since HFE is known to regulate iron uptake, we studied how modulating the iron status of GBM cells impacted their ability to migrate and adhere. We found that increasing the iron pool of these mouse glioma models by exposure to exogenous iron compounds decreased migratory capacity. To better understand mechanistically how HFE and iron status impacted migration and adhesion, we probed how expression of integrins and their downstream signaling molecules, the Rho GTPases were altered in response to iron. We found that exposure to iron decreased levels of the Rho GTPases Cdc42 and RhoA. Furthermore, cells that overexpressed HFE were found to have increased expression of integrin β1 and integrin α5 suggesting that HFE and iron may impact integrins and their downstream signaling pathways to alter migration of GBM cells.
TAMI-44. ASSOCIATION OF CIRCADIAN CLOCK GENE EXPRESSION WITH GLIOMA TUMOR MICROENVIRONMENT AND PATIENT SURVIVAL
Authors: De La Cruz Minyety Julianie , Shuboni-Mulligan Dorela , Briceno Nicole , Young Jr. Demarrius , Gilbert Mark , Celiku Orieta , Armstrong Terri
Published: November 2021

Circadian clock genes have been linked to differences in clinical outcomes in cancer, including gliomas. However, these studies have not accounted for established prognostic markers, including mutations in Isocitrate Dehydrogenase (IDH). To study the connection between circadian clock genes and glioma outcomes while accounting for IDH mutational status, we analyzed multiple publicly available gene expression datasets. Unsupervised clustering of 13 clock gene transcriptomic signatures from The Cancer Genome Atlas resulted in four distinct transcriptomic clusters, two clusters were enriched for IDH mutant (Circadian 1-2) and the others for IDH wild-type gliomas (Circadian 3-4). Within these clusters we observed differential prognosis of the patients by Kaplan–Meier analysis (Circadian 1-2, p=0.0001; Circadian 3-4, p=0.0002) suggesting that these transcriptomic circadian subtypes might reflect different disease states. Further analyses using Cox Proportional Hazards Regression showed that lower Period (PER) gene expression was associated with worse prognosis (increasing PER expression HR=0.655, p=0.007) independent of IDH wild-type status (HR=5.312, p< 0.001) and increasing age (HR = 1.04, p< 0.001). Lower PER expression was associated with enrichment of a number of immune signaling pathways. These findings prompted the exploration of the relationship between microenvironment and clock genes using the Ivy GAP dataset to explore tumor location-specific differences and single cell RNA sequencing data from Darmanis (accession: GSE84465) to explore cell-specific differences. Circadian clock genes were found to be differentially expressed across anatomical tumor locations and cell types, including microglia. In ongoing studies we are examining the role of the microenvironment and PER2 expression on tumor growth by disrupting PER2 expression in tumor cells and microglia using IDH mutant and wild-type in vitro models. Clock gene expression is a potential prognostic biomarker in glioma and further studies to elucidate the importance of circadian rhythms in other cell types beyond the tumor are warranted.
TAMI-48. THE KETOGENIC DIET IS INEFFECTIVE IN PRECLINICAL MODELS OF IDH1 WILD-TYPE AND IDH1 MUTANT GLIOMA
Authors: Javier Rodrigo , Horbinski Craig
Published: November 2021

Despite decades of intensive research, infiltrative gliomas are still usually lethal and challenging to treat. A subset of gliomas contains mutations in isocitrate dehydrogenase 1 (IDH1mut), which disrupts cellular biochemistry; such gliomas are generally less aggressive than their IDH1 wild-type (IDH1wt) counterparts. Some preclinical studies have suggested that a ketogenic diet (KD), characterized by low-carbohydrate and high-fat content, may be beneficial against a variety of cancers, including gliomas. However, not all studies have shown promising results, and to date, no study has addressed the sensitivity of glioma cells to KD in the specific context of the endogenous IDH1mut metabolic landscape. The aim of the current study was to compare the effects of KD in preclinical models to IDH1wt versus IDH1mut gliomas. In vitro treatment of patient-derived IDH1wt and IDH1mut glioma cells with the ketone body β-hydroxybutyrate showed no significant effect on cell proliferation in a low glucose culture environment. Likewise, the in vivo flank growth rates of these patient-derived IDH1wt and IDH1mut glioma xenografts showed no significant difference when mice were fed KD versus regular diet (GBM12 p=0.98, GBM164 p=0.4, GBM196 p=0.11). Finally, KD had no effect on the survival of mice engrafted with isogenic Sleeping-Beauty transposase-engineered IDH1wt (median control survival 22 days versus treatment 23 days, p=0.23) or IDH1mut glioma cells (median control survival 26.5 days versus treatment 26 days, p=0.81). These data suggest that IDH1mut gliomas are not more responsive than IDH1wt gliomas to KD, and that clinical trials further exploring KD in this subset of glioma patients are probably not warranted.
TAMI-49. JAK STAT INHIBITION REVERSES MYELOID CELL INDUCED ANTI TUMOR IMMUNITY IN T CELLS
Authors: Ravi Vidyha , Neidert Nicolas , Will Paulina , Joseph Kevin , Hofmann Ulrich , Beck Jürgen , Schnell Oliver , Heiland Dieter Henrik
Published: November 2021

BACKGROUND

Many central questions about the immunosuppressive microenvironment in glioblastoma (GBM) remain unanswered, particularly the interaction with lymphoid and myeloid populations. Here, we combined single-cell (scRNA) and spatial transcriptomics (stRNA) to comprehensively characterize the immune interaction with GBM.

MATERIAL AND METHODS

We performed scRNA-Seq of 50k CD45+ cells (8 patients) and inferred transcriptional programs and fate decisions in T cells. A novel algorithm (Nearest functionally connected neighbor) was used to predict interacting cells, further validated using spatial transcriptomics and immunofluorescence. Our findings were validated in our human neocortical glioblastoma model with autografted T cells.

RESULTS

Integration of st/scRNA-seq revealed a transcriptional shift of T cells towards exhaustion/hypoxia induced dysfunction. Pseudo-time analysis revealed increased Interleukin 10 (IL10) response during the Tcell transformation from the effector to the exhausted state. Using NFCN we identified a subset of HMOX1+ myeloid cells (STAT/HMOX axis), responsible for this IL10 release. Computational findings were validated using our human neocortical glioblastoma model with autografted T cells, where IL10R-inhibition/myeloid cell depletion prevented T cell exhaustion/dysfunction (p < 0.01) . In order to target the STAT3/HMOX1 axis we used a JAK/STAT inhibitor in our model which showed a drastic reduction of IL10 release (p< 0.02) and concordant activation of T cells. Clinically, one patient treated with a JAK/STAT-inhibitor in a neoadjuvant setting, 4 weeks prior to the recurrent GBM surgery, led to a significant increase (p< 0.001) in effector T cell population.

CONCLUSION

Our findings suggest that targeting the myeloid compartment of GBM provides an opportunity to convert a “cold” into “hot” immune environment which might be helpful to improve all T cell based therapies in the future.
TAMI-51. HORIZONTAL MITOCHONDRIAL TRANSFER FROM THE TUMOR MICROENVIRONMENT TO GLIOBLASTOMA INCREASES TUMORIGENICITY
Authors: Watson Dionysios , Bayik Defne , Lathia Justin
Published: November 2021

Communication between glioblastoma (GBM) and its microenvironment facilitates tumor growth and therapeutic resistance, and is facilitated through a variety of mechanisms. Organelle transfer between cells was recently observed, including mitochondria transfer from astrocytes to neurons after ischemic stroke. Given the dependence of GBM on microenvironmental interactions, we hypothesized that mitochondria transfer from tumor microenvironment to GBM cells could occur and affect metabolism and tumorigenicity. We interrogated this in vivo by establishing intracranial GBM tumors in mito::mKate2 mice (with trackable fluorescent mitochondria) using syngeneic GFP-expressing tumor cells (SB28 and GL261 models). We also cultured stromal cell types from mito::mKate2 mice with tumor cells, enabling sorting of tumor cells with and without exogenous mitochondria. Confocal microscopy revealed horizontal transfer of mKate2+ mitochondria from mouse cells to implanted GBM cells in vivo and was confirmed by flow cytometry where 20-40% of GBM cells acquired exogenous mitochondria. Transfer was negligible in wildtype mice transplanted with mito::mKate2 bone marrow cells, suggesting that brain-resident cells were the main donors. In vitro, astrocytes and microglia exhibited 5 to 10-fold higher mitochondrial transfer rate than bone-marrow derived macrophages. Seahorse metabolic profiling revealed that GBM cells with mKate2+ mitochondria had 40% lower respiratory reserve compared to cells without exogenous mitochondria. Median survival of mice implanted with SB28 that acquired mitochondria was significantly shorter and in vivo limiting dilution confirmed the frequency of tumor-initiating cells was 3-fold higher in SB28 cells with exogenous mitochondria. Our data indicate that horizontal mitochondrial transfer from brain-resident glia to mouse GBM tumors alters tumor cell metabolism and increases their tumorigenicity. Ongoing studies are assessing gene expression in GBM cells acquiring exogenous mitochondria; validating findings in human specimens; and screening for transfer inhibitor drugs. Horizontal mitochondrial transfer represents a foundational tumor microenvironment interaction contributing to glioblastoma plasticity, and is likely to inform next-generation treatment strategies.
TAMI-52. NEURONAL MECHANISMS OF BRAIN TUMOR INVASION
Authors: Venkataramani Varun , Yang Yvonne , Schubert Marc , Beretta Carlo , Botz Michael , Fankhauser Laura , Tetzlaff Svenja , Wagner Julia , Garofano Luciano , Freudenberg Alexander , Slowik Martin , Tanev Dimitar , Döring Leif , Iavarone Antonio , Wick Wolfgang , Kuner Thomas , Winkler Frank
Published: November 2021

Incurable gliomas are characterized by their infiltration into the whole brain. Recently, we described tumor microtubes as a novel structure contributing to glioma cell invasion and uncovered synaptic contacts on glioma cells that drive brain tumour progression. However, the exact effects of neuronal activity on glioma cell motility are yet unclear. Here, we show how a recently described neuronal-like cellular transcription state of glioblastoma cells is correlated to glioma cell invasion in vivo. To unravel the details of neuronal features of glioma invasion in space and time, we established a novel approach of intravital imaging for brain tumor cells with a membrane-bound GFP combined with deep learning algorithms that are used to track glioma cell processes with a high temporal resolution over several hours. This approach uncovers how invading tumor microtubes use Levy-like movement patterns indicative of efficient search patterns often employed by animal predators searching for scarce resources such as food. Neuronal activity is able to accelerate the tumor microtube dynamics, accelerate the Levy-like movement patterns and increase the overall invasion speed of glioma cells. These processes are mediated by local calcium transients in glioma cell somata and tumor microtubes. In accordance, genetic manipulation and pharmacological perturbation of AMPA receptors reduces tumor microtube length, number and branching points by interfering with intracellular calcium transients. All in all, the work here uncovers novel neuronal activity-mediated mechanisms of glioma cell invasion, a hallmark of this yet fatal disease.
TAMI-60. MODULATION OF CELL BIOMECHANICS THROUGH GUIDANCE RECEPTOR PLEXIN-B2 FACILITATES GLIOBLASTOMA INFILTRATION
Authors: Friedel Roland , Huang Yong , Tejero Rut , Alves Chrystian Junqueira , Brusco Concetta , Hannah Theodore , Lee Vivian , Dai Guohao , Zou Hongyan
Published: November 2021

Infiltrative growth is a major cause of the high lethality of malignant brain tumors such as glioblastoma (GBM). The study of the contribution of biomechanical processes to GBM invasion is an emerging field. We show here that GBM cells upregulate the guidance receptor Plexin-B2 to gain invasiveness by modulating their biomechanical properties. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies have established Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness. Ongoing investigations focus on the regulation of the biomechanical properties of cell membrane and cell actomyosin cortex through plexins that provide GBM cells with the mechanical dynamics to penetrate to restricted space.
TAMI-64. IMMUNE ENVIRONMENT SHAPES GLIOBLASTOMA HETEROGENEITY IN TIME AND SPACE
Authors: Will Paulina , Kückelhaus Jan , Benotmane Jasim Kada , Joseph Kevin , Beck Jürgen , Schnell Oliver , Ravi Vidyha , Heiland Dieter Henrik
Published: November 2021

Glioblastomas are embedded into an immunosuppressive microenvironment resulting in limited success of immunotherapies. Although, we and others observed defined myeloid-tumor crosslinks reducing T cell homing and activation, the spatial context of these interactions remained unexplored. Here, we provide evidence, that local T cell infiltration results in a defined activation of myeloid cells causing transcriptional reprogramming of tumor cells reminiscent of reactive transformation in mature astrocytes. Through integration of spatially resolved transcriptomics and imaging mass cytometry (n=18, 39 protein glioma panel) we mapped defined transcriptional responses in areas of high or low T cell infiltration respectively. Functional analysis revealed that areas of large T cell infiltration are enriched for glial (CHI3L1, GFAP and VIM) and inflammatory genes (HLA-DRA, C3, CCL4, CCL3). We found that marker genes of common reactive states in mature astrocytes significantly overlap with the reactive immune program of glioblastoma cells (f-score 0.76, p=2.2e-10). Increased numbers of CD163+ cells were found in surrounding areas of T cell infiltration and spatially linked to defined immunosuppressive release of IL10, recently reported as a major driver of T cell exhaustion. To support our findings, we injected a primary glioblastoma cell line into cortex slices of three different human and rodent donors. After 7 days of tumor growth, we performed scRNA-sequencing of FACS-sorted tumor cells and baseline cell culture cells. Compared to baseline, we found cells of all reported transcriptional states, confirming the dynamic adaptation of cells within a neural environment. In elderly donors, a significant accumulation of reactive immune programs (ANOVA p< 0.001) was observed. Immunostainings confirmed an increased myeloid cell activation in these donors. Our data suggest that inflammatory stimuli in the glioblastoma microenvironment cause transcriptional reprogramming in glioblastoma similar to inflammatory transformation of reactive astrocytes. The spatial exclusivity of these programs highlights the value of a spatial perspective on heterogeneity.
TAMI-65. FASN-MEDIATED LIPID SYNTHESIS HAMPERS ANTI-CANCER IMMUNITY OF GLIOBLASTOMA
Authors: De Martino Mara , Daviaud Camille , Vanpouille-Box Claire
Published: November 2021

Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-cancer immunity by at least generating type I interferon (IFN-I) responses. However, RT also exacerbates potent immune inhibitory mechanisms that facilitate immune evasion. Notably, increased lipid synthesis by the fatty acid synthase (FASN) is an emerging mechanism that can account for the deceiving treatment efficacy and immune escape of GBM. Therefore, we hypothesize that FASN-mediated lipid synthesis represents an innate immune evasion mechanism of irradiated GBM. Supporting this hypothesis, we observed that 10 gray (Gy) irradiation of murine GBM cell lines, GL261 and CT2A, upregulates FASN protein expression and increase cellular lipid content determined by BODIPY staining and electronic microscopy. Interestingly, this effect was abrogated when GBM cells were incubated with an inhibitor of FASN. Next, to ask whether FASN was impairing RT-induced IFN-I, GL261 and CT2A cells were engineered to express an inducible shRNA silencing FASN (GBMshFASN) or its non-silencing control (GBMshNS). Irradiation of GBMshNS cells enhanced the secretion of IFN-beta and CXCL10, but this effect was more pronounced when FASN was blocked. Finally, GBMshNS and GBMshFASN cells were orthotopically implanted in mice on day 0. On day 10, selective irradiation (10Gy) was performed to the tumor. Tumor growth and immune contexture were evaluated on day 17. Magnetic resonance imaging revealed that FASN knockdown reduces tumor growth independently from RT. However, in situ immunofluorescence of GBMshFASN tumors showed increased infiltration of CD8+ T cells and CD11c+ cells only in irradiated mice bearing GBMshFASN tumors. Altogether, our data suggest that RT rewires the energy supply of GBM by promoting FASN-mediated lipid synthesis to foster immune evasion. Targeting FASN is a promising strategy to promote anti-cancer immunity and sensitize irradiated GBM to immunotherapies.
TAMI-66. FUNCTIONAL ALTERATIONS IN CORTICAL PROCESSING OF SPEECH IN GLIOMA-INFILTRATED CORTEX
Authors: Aabedi alexander , Lipkin Benjamin , Kaur Jasleen , Kakaizada Sofia , Young Jacob , Lee Anthony , Krishna Saritha , Chang Edward , Brang David , Hervey-Jumper Shawn
Published: November 2021

INTRODUCTION

Recent developments in the biology of malignant gliomas have demonstrated that glioma cells interact with neurons through both paracrine signaling and electrochemical synapses. Glioma-neuron interactions consequently modulate the excitability of local neuronal circuits, and it is unclear the extent to which glioma-infiltrated cortex can meaningfully participate in neural computations. For example, gliomas may result in a local disorganization of activity that impedes the transient synchronization of neural oscillations. Alternatively, glioma-infiltrated cortex may retain the ability to engage in synchronized activity, in a manner similar to normal-appearing cortex, but exhibit other altered spatiotemporal patterns of activity with subsequent impact on cognitive processing.

METHODS

Here, we acquired invasive electrophysiologic recordings to sample both normal-appearing and glioma-infiltrated cortex during speech initiation in order to measure language task-related circuit dynamics of IDH-wild-type glioblastoma patients. We then applied an information theoretical framework to directly compare the encoding capacity and decodability of signals arising from these regions.

RESULTS

We find that glioma-infiltrated cortex engages in synchronous activity during task performance in a manner similar to normal-appearing cortex, but recruits a diffuse spatial network. On a temporal scale, we show that glioma-infiltrated cortex has lower capacity for information encoding when performing nuanced tasks such as speech production of monosyllabic versus polysyllabic words. As a result, temporal decoding strategies for distinguishing monosyllabic from polysyllabic words were feasible for signals arising from normal-appearing cortex, but not from glioma-infiltrated cortex.

CONCLUSION

These findings inform our understanding of cognitive processing in patients with malignant gliomas and have implications for patient survival, neuromodulation, and prosthetics in patients with malignant gliomas.
TAMI-70. METABOLIC VULNERABILITY TO GPX4 INHIBITION AND FERROPTOSIS OF QUIESCENT ASTROCYTE-LIKE GLIOMA CELL POPULATIONS
Authors: Banu Matei , Dovas Athanassios , Argenziano Michael , Zhao Wenting , Higgins Dominique , Upadhyayula Pavan , Mahajan Aayushi , Humala Nelson , Nguyen Trang , Zandkarimi Fereshteh , Siegelin Markus D , Brent Stockwell , Sims Peter , Bruce Jeffrey N , Canoll Peter
Published: November 2021

Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.
TAMI-77. CHARACTERIZATION OF METABOLIC DIFFERENCES IN MENINGIOMAS USING TUMOR TISSUE AND PATIENT-DERIVED CELL LINES
Authors: Rabow Zach , Morningstar Taryn , Fiehn Oliver , Lechpammer Mirna
Published: November 2021

As more information emerges on metabolic changes in brain pathologies, mass spectrometry methodologies are needed to investigate cellular changes in primary brain tumors. Many approaches use technologies that lack sensitivity and selectivity, which hinders discovering potential novel diagnostic and prognostic features. Fresh frozen tissue from 26 patients (57% women, 43% men, age 21-67) underwent surgical resection for newly diagnosed meningiomas. We used patient-derived cell lines and arachnoid cells to validate and compare metabolic differences in patient tumor tissue. Primary metabolism (GC-TOF), lipidomics, and biogenic amines (RPLC and HILIC-HRMS) were analyzed using 10 mg of tissue and 1x106 cells. RESULTS: Primary metabolism and lipidomics showed significant differences between Grade I and Grade II tumors. We identified over one thousand total metabolites through authentic standards and MS2 fragmentation patterns. Ascorbic Acid was enriched in Grade I tumors compared to Grade II and enriched in tumors compared to non-neoplastic dura tissue from the same patient. Lysosomal activation in atypical meningiomas is indicated by a 2-fold increase (compared to Grade I tumors) of Bis(monoacylglycero)phosphate metabolites. We grouped metabolites into fifteen classes based on chemical ontology and function. Statistically significant differences were observed in total amino acids (AA), basic AA, cyclic AA, sulfur-containing AA, branched-chain AA, dipeptides, histidine-containing dipeptides, vitamins and cofactors, glutathione metabolites, acylcarnitine’s, sphingomyelins, phosphatidylethanolamines, phosphatidylinositol’s, cardiolipins, and nucleic acids. Thymine-containing nucleic acids and biogenic monoamines were 2-fold higher in Grade I compared to Grade II tumors. Using novel untargeted metabolomics, we found multiple classes of metabolites that were enriched in Grade II meningiomas compared to Grade I. This points towards possible pathways that drive malignancy and biomarkers that could be useful for diagnosis, grading, and treatment selection.
TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG
Authors: Yadav Viveka Nand , Harris Micah K , Messinger Dana , Thomas Chase , Cummings Jessica R , Yang Tao , Woo Rinette , Siddaway Robert , Burkert Martin , Stallard Stefanie , Qin Tingting , Mullan Brendan , Siada Ruby , Ravindran Ramya , Niculcea Michael , Ginn Kevin F , Gener Melissa A H , Dorris Kathleen , Vitanza Nicholas A , Schmidt Susanne V , Spitzer Jasper , Li Jiang , Filbin Mariella G , Cao Xuhong , Castro Maria G , Lowenstein Pedro R , Mody Rajen , Chinnaiyan Arul , Desprez Pierre , McAllister Sean , Hawkins Cynthia , Waszak Sebastian M , Venneti Sriram , Koschmann Carl
Published: November 2021

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.
TMOD-03. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION
Authors: Taisto Mandy , Tschida Barbara , Coutts Alexander , Campbell Jarryd , Corbiere Stanislas , Duerre Dylan , Watson Adrienne
Published: November 2021

Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is desperately needed, as the standard of care universally fails to cure patients and the 5-year survival rate remains extremely low. GBM therapeutic development is hampered by the lack of relevant preclinical models for preclinical studies. To mitigate this problem, we have developed a genetic model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain in vivo by direct injection of gene delivery reagents to the lateral ventricle, altering major signaling pathways frequently changed in human GBM. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM. We have altered six major human GBM-associated signaling pathways and modeled molecular GBM subclasses. We have also engineered a secreted tumor reporter that can be used to monitor tumor size through a simple blood test. This somatic cell gene-modification platform we have developed in the minipig allows us to reproduce the genetic heterogeneity seen in GBM and understand the impact of the tumor microenvironment, immune system, and response to therapy. This minipig model of GBM Is being used to test the standard of care against novel therapies in preclinical studies, and biopsy, surgical, imaging, and radiation therapy techniques are being optimized in this mode to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health though SBIR grant # 1R43CA235837-01A1.