Abstract INTRODUCTION Glioblastoma (GBM) is the most common brain tumor, however, is a rare occurrence in children and is poorly characterized. We evaluated the characteristics and outcomes of pediatric GBM (pGBM). METHODS Retrospective analysis of pediatric (age< 18) patients diagnosed with GBM undergoing first glioblastoma resection at our brain tumor center (2005- 2016). RESULTS From 1457 GBM patients, we identified twenty-four (1.65%) pGBMs (Median Age=9 years, Females=45.8%). Median overall survival (OS) was 32.1 months, while the median progression-free survival was 11.5 months. The commonest symptoms at presentation were headaches (54.2%,n=13) and motor symptoms (50%,n=12). Mean tumor diameter was 4.5 cm and 25% of the cohort underwent gross total resection (GTR) of their tumor. Univariate analysis revealed median OS significantly associated with tumor extent of resection (GTR=56.4 months; STR/Biopsy=13.7 months, p=0.001), age at surgery (>10 years=43.9 months, < 10 years= 17.2 months, p=0.01), tumor size (> 4cm= 9.1 months, < 4cm=56.9 months, p=0.01),motor symptoms at presentation (present=14.9 months, absent=41.04 months, p=0.02) and infratentorial tumors (infratentorial=17.4 vs supratentorial=53.4 months, p=0.02). Multivariate analysis revealed GTR (HR 0.2[95% CI 0.07–0.72]; p=0.03), Age >10 years (HR 0.6[95% CI 0.02–0.64]; p=0.002), tumor >4 cm (HR 2.89[95% CI 1.88–4.11]; p=0.001) and EGFR amplification (HR 3.48[95% CI 0.82–17.4]; p=0.005) to be independent predictors of OS. Comparing patients under and over 10 years, we found that older patients had smaller tumors at presentation (4.9 vs 3.6 cms, p=0.03), greater rates of preoperative temozolomide (n=1,7.7% vs n=6, 54.5%) and bevacizumab (n=1,7.7% vs n=4, 36.4%) treatment, and lower rates of EGFR amplification (66.7% vs 11.1%) that could explain survival disparities between groups. CONCLUSION Motor symptoms, larger tumors at presentation and tumor EGFR amplification may be indictive of poorer outcomes in pGBM. However, maximal tumor resection, aggressive chemoradiation and tumor presentation at age >10 years may confer better prognosis in these patients.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy. SIGNIFICANCE: Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1498/F1.large.jpg.