Background Glioblastoma (GBM), a highly malignant brain tumor, invariably recurs after therapy. Quiescent GBM cells represent a potential source of tumor recurrence, but little is known about their molecular underpinnings. Methods Patient-derived GBM cells were engineered by CRISPR/Cas9-assisted knock-in of an inducible histone2B-GFP (iH2B-GFP) reporter to track cell division history. We utilized an in vitro 3D GBM organoid approach to isolate live quiescent GBM (qGBM) cells and their proliferative counterparts (pGBM) to compare stem cell properties and therapy resistance. Gene expression programs of qGBM and pGBM cells were analyzed by RNA-Seq and NanoString platforms. Findings H2B-GFP-retaining qGBM cells exhibited comparable self-renewal capacity but higher therapy resistance relative to pGBM. Quiescent GBM cells expressed distinct gene programs that affect cell cycle control, metabolic adaptation, and extracellular matrix (ECM) interactions. Transcriptome analysis also revealed a mesenchymal shift in qGBM cells of both proneural and mesenchymal GBM subtypes. Bioinformatic analyses and functional assays in GBM organoids established hypoxia and TGFβ signaling as potential niche factors that promote quiescence in GBM. Finally, network co-expression analysis of TCGA glioma patient data identified gene modules that are enriched for qGBM signatures and also associated with survival rate. Interpretation Our in vitro study in 3D GBM organoids supports the presence of a quiescent cell population that displays self-renewal capacity, high therapy resistance, and mesenchymal gene signatures. It also sheds light on how GBM cells may acquire and maintain quiescence through ECM organization and interaction with niche factors such as TGFβ and hypoxia. Our findings provide a starting point for developing strategies to tackle the quiescent population of GBM. Fund National Institutes of Health (NIH) and Deutsche Forschungsgemeinschaft (DFG).

The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, here we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR, we validate TEAD1 trans occupancy at accessibility sites within AQP4, EGFR, and CDH4. To further characterize TEAD’s functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1–AQP4 in GBM migration.

Many focal cerebral ischemia models utilize the middle cerebral artery occlusion (MCAO) evoked by coagulation to induce ischemic damage in the cortex and mimic the pathology observed in human patients. A second, increasingly popular model, the photothrombotic stroke, uses a laser beam to irradiate the MCA after administration of a photosensitizing dye. This widely used procedure is slowly replacing the MCAO model because of the easiness of the surgical protocol and the reproducibility of the damage. However, the photochemical reaction also results in wider microvascular injury. In this study, we have evaluated the impact of these two types of stroke in the cell survival and evolution of stroke, focusing on microglial cells, the first responders to cell injury. Two groups of heterozygote Cx3CR1-GFP reporter mice (to follow microglia) were subject to stroke injury either with coagulator-mediated occlusion or photothrombotic MCA damage. Microglial cells dynamics of activation and phagocytosis together with astrocytic response and leukocyte infiltration were characterized at 1, 3 and 7 days after damage. Photothrombotic stroke delayed microglial and astrocytic invasion of the ischemic core and accumulation of phagocytic microglia. It also elicited higher levels of inflammatory cytokines/chemokines and increased infiltration from the periphery. In addition, only the neurons in the MCAO stroke showed phenotype plasticity by downregulating the transcription factor NeuN. These data provide a better understanding of the exact temporal and spatial dynamics of the inflammatory response in these two animal models of stroke and identify more relevant targets for human therapy.

SummaryCharacterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR+/− populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand (LBEGFR+), and uniquely compared their functional and molecular properties. LBEGFR+ populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LBEGFR+ GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR+ populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology.