Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer’s cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.

Multiple sclerosis is a chronic inflammatory disease of the CNS 1 . Astrocytes contribute to the pathogenesis of multiple sclerosis 2 , but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Characterizing the developmental trajectory of oligodendrocyte progenitor cells (OPC) is of great interest given the importance of these cells in the remyelination process. However, studies of human OPC development remain limited by the availability of whole cell samples and material that encompasses a wide age range, including time of peak myelination. In this study, we apply single cell RNA sequencing to viable whole cells across the age span and link transcriptomic signatures of oligodendrocyte‐lineage cells with stage‐specific functional properties. Cells were isolated from surgical tissue samples of second‐trimester fetal, 2‐year‐old pediatric, 13‐year‐old adolescent, and adult donors by mechanical and enzymatic digestion, followed by percoll gradient centrifugation. Gene expression was analyzed using droplet‐based RNA sequencing (10X Chromium). Louvain clustering analysis identified three distinct cellular subpopulations based on 5,613 genes, comprised of an early OPC (e‐OPC) group, a late OPC group (l‐OPC), and a mature OL (MOL) group. Gene ontology terms enriched for e‐OPCs included cell cycle and development, for l‐OPCs included extracellular matrix and cell adhesion, and for MOLs included myelination and cytoskeleton. The e‐OPCs were mostly confined to the premyelinating fetal group, and the l‐OPCs were most highly represented in the pediatric age group, corresponding to the peak age of myelination. Cells expressing a signature characteristic of l‐OPCs were identified in the adult brain in situ using RNAScope. These findings highlight the transcriptomic variability in OL‐lineage cells before, during, and after peak myelination and contribute to identifying novel pathways required to achieve remyelination.

These results provide additional information to guide healthcare professionals in predicting overall outcome acutely post-TBI.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

The objective of this study was to examine the performance of patients with traumatic brain injury (TBI) on the Montreal Cognitive Assessment (MoCA). The MoCA was administered to 214 patients with TBI during their acute care hospitalization in a Level 1 trauma center. The results showed that patients with severe TBI had lower scores on the MoCA compared with patients with mild and moderate TBI, F(2, 211) = 10.35, p = .0001. This difference was found for visuospatial/executive, attention, and orientation subtests (p < .05). Linear regression demonstrated that age, education, TBI severity, and the presence of neurological antecedents were the best predictors of cognitive impairments explaining 42% of the total variability of the MoCA. This information can enable clinicians to predict early cognitive impairments and plan cognitive rehabilitation earlier in the recovery process.

Myelin destruction and oligodendrocyte (OL) death consequent to metabolic stress is a feature of CNS disorders across the age spectrum. Using cells derived from surgically resected tissue, we demonstrate that young (<age 5) pediatric-aged sample OLs are more resistant to in-vitro metabolic injury than fetal O4+ progenitor cells, but more susceptible to cell death and apoptosis than adult-derived OLs. Pediatric but not adult OLs show measurable levels of TUNEL+ cells, a feature of the fetal cell response. The ratio of anti- vs pro-apoptotic BCL-2 family genes are increased in adult vs pediatric (<age 5) mature OLs and in more mature OL lineage cells. Lysosomal gene expression was increased in adult and pediatric compared to fetal OL lineage cells. Cell death of OLs was increased by inhibiting pro-apoptotic BCL-2 gene and autophagy activity. These distinct age-related injury responses should be considered in designing therapies aimed at reducing myelin injury.

Background Sixty percent of surgically resected brain metastases (BrM) recur within 1 year. These recurrences have long been thought to result from the dispersion of cancer cells during surgery. We tested the alternative hypothesis that invasion of cancer cells into the adjacent brain plays a significant role in local recurrence and shortened overall survival. Methods We determined the invasion pattern of 164 surgically resected BrM and correlated with local recurrence and overall survival. We performed single-cell RNA sequencing (scRNAseq) of &gt;15,000 cells from BrM and adjacent brain tissue. Validation of targets was performed with a novel cohort of BrM patient-derived xenografts (PDX) and patient tissues. Results We demonstrate that invasion of metastatic cancer cells into the adjacent brain is associated with local recurrence and shortened overall survival. scRNAseq of paired tumor and adjacent brain samples confirmed the existence of invasive cancer cells in the tumor-adjacent brain. Analysis of these cells identified cold-inducible RNA-binding protein (CIRBP) overexpression in invasive cancer cells compared to cancer cells located within the metastases. Applying PDX models that recapitulate the invasion pattern observed in patients, we show that CIRBP is overexpressed in highly invasive BrM and is required for efficient invasive growth in the brain. Conclusions These data demonstrate peritumoral invasion as a driver of treatment failure in BrM that is functionally mediated by CIRBP. These findings improve our understanding of the biology underlying postoperative treatment failure and lay the groundwork for rational clinical trial development based upon invasion pattern in surgically resected BrM.