MAFG-driven astrocytes promote CNS inflammation.

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, 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.

Developmental trajectory of oligodendrocyte progenitor cells in the human brain revealed by single cell RNA sequencing.

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.

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease.

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples.

Programs, Origins, and Niches of Immunomodulatory Myeloid Cells in Gliomas.

Gliomas are incurable malignancies notable for an immunosuppressive microenvironment with abundant myeloid cells whose immunomodulatory properties remain poorly defined. Here, utilizing scRNA-seq data for 183,062 myeloid cells from 85 human tumors, we discover that nearly all glioma-associated myeloid cells express at least one of four immunomodulatory activity programs: Scavenger Immunosuppressive, C1Q Immunosuppressive, CXCR4 Inflammatory, and IL1B Inflammatory. All four programs are present in IDH1 mutant and wild-type gliomas and are expressed in macrophages, monocytes, and microglia whether of blood or resident myeloid cell origins. Integrating our scRNA-seq data with mitochondrial DNA-based lineage tracing, spatial transcriptomics, and organoid explant systems that model peripheral monocyte infiltration, we show that these programs are driven by microenvironmental cues and therapies rather than myeloid cell type, origin, or mutation status. The C1Q Immunosuppressive program is driven by routinely administered dexamethasone. The Scavenger Immunosuppressive program includes ligands with established roles in T-cell suppression, is induced in hypoxic regions, and is associated with immunotherapy resistance. Both immunosuppressive programs are less prevalent in lower-grade gliomas, which are instead enriched for the CXCR4 Inflammatory program. Our study provides a framework to understand immunomodulatory myeloid cells in glioma, and a foundation to develop more effective immunotherapies.

Single-Cell Multiomics Reveals Clonal T-Cell Expansions and Exhaustion in Blastic Plasmacytoid Dendritic Cell Neoplasm.

The immune system represents a major barrier to cancer progression, driving the evolution of immunoregulatory interactions between malignant cells and T-cells in the tumor environment. Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare acute leukemia with plasmacytoid dendritic cell (pDC) differentiation, provides a unique opportunity to study these interactions. pDCs are key producers of interferon alpha (IFNA) that play an important role in T-cell activation at the interface between the innate and adaptive immune system. To assess how uncontrolled proliferation of malignant BPDCN cells affects the tumor environment, we catalog immune cell heterogeneity in the bone marrow (BM) of five healthy controls and five BPDCN patients by analyzing 52,803 single-cell transcriptomes, including 18,779 T-cells. We test computational techniques for robust cell type classification and find that T-cells in BPDCN patients consistently upregulate interferon alpha (IFNA) response and downregulate tumor necrosis factor alpha (TNFA) pathways. Integrating transcriptional data with T-cell receptor sequencing via shared barcodes reveals significant T-cell exhaustion in BPDCN that is positively correlated with T-cell clonotype expansion. By highlighting new mechanisms of T-cell exhaustion and immune evasion in BPDCN, our results demonstrate the value of single-cell multiomics to understand immune cell interactions in the tumor environment.

Glioblastoma scRNA-seq shows treatment-induced, immune-dependent increase in mesenchymal cancer cells and structural variants in distal neural stem cells.

BACKGROUND: Glioblastoma is a treatment-resistant brain cancer. Its hierarchical cellular nature and its tumor microenvironment (TME) before, during, and after treatments remain unresolved. METHODS: Here, we used single-cell RNA sequencing to analyze new and recurrent glioblastoma and the nearby subventricular zone (SVZ). RESULTS: We found 4 glioblastoma neural lineages are present in new and recurrent glioblastoma with an enrichment of the cancer mesenchymal lineage, immune cells, and reactive astrocytes in early recurrences. Cancer lineages were hierarchically organized around cycling oligodendrocytic and astrocytic progenitors that are transcriptomically similar but distinct to SVZ neural stem cells (NSCs). Furthermore, NSCs from the SVZ of patients with glioblastoma harbored glioblastoma chromosomal anomalies. Lastly, mesenchymal cancer cells and TME reactive astrocytes shared similar gene signatures which were induced by radiotherapy in a myeloid-dependent fashion in vivo. CONCLUSION: These data reveal the dynamic, immune-dependent nature of glioblastoma's response to treatments and identify distant NSCs as likely cells of origin.

Extent of Tumor Resection and Survival in Pediatric Patients With High-Grade Gliomas: A Systematic Review and Meta-analysis.

Importance: Pediatric patients with high-grade gliomas have a poor prognosis. The association among the extent of resection, tumor location, and survival in these patients remains unclear. Objective: To ascertain whether gross total resection (GTR) in hemispheric, midline, or infratentorial pediatric high-grade gliomas (pHGGs) is independently associated with survival differences compared with subtotal resection (STR) and biopsy at 1 year and 2 years after tumor resection. Data Sources: PubMed, EBMR, Embase, and MEDLINE were systematically reviewed from inception to June 3, 2022, using the keywords high-grade glioma, pediatric, and surgery. No period or language restrictions were applied. Study Selection: Randomized clinical trials and cohort studies of pHGGs that stratified patients by extent of resection and reported postoperative survival were included for study-level and individual patient data meta-analyses. Data Extraction and Synthesis: Study characteristics and mortality rates were extracted from each article. Relative risk ratios (RRs) were pooled using random-effects models. Individual patient data were evaluated using multivariate mixed-effects Cox proportional hazards regression modeling. The PRISMA reporting guideline was followed, and the study was registered a priori. Main Outcomes and Measures: Hazard ratios (HRs) and RRs were extracted to indicate associations among extent of resection, 1-year and 2-year postoperative mortality, and overall survival. Results: A total of 37 studies with 1387 unique patients with pHGGs were included. In study-level meta-analysis, GTR had a lower mortality risk than STR at 1 year (RR, 0.69; 95% CI, 0.56-0.83; P < .001) and 2 years (RR, 0.74; 95% CI, 0.67-0.83; P < .001) after tumor resection. Subtotal resection was not associated with differential survival compared with biopsy at 1 year (RR, 0.82; 95% CI, 0.66-1.01; P = .07) but had decreased mortality risk at 2 years (RR, 0.89; 95% CI, 0.82-0.97; P = .01). The individual patient data meta-analysis of 27 articles included 427 patients (mean [SD] age at diagnosis, 9.3 [5.9] years), most of whom were boys (169 of 317 [53.3%]), had grade IV tumors (246 of 427 [57.7%]), and/or had tumors that were localized to either the cerebral hemispheres (133 of 349 [38.1%]) or midline structures (132 of 349 [37.8%]). In the multivariate Cox proportional hazards regression model, STR (HR, 1.91; 95% CI, 1.34-2.74; P < .001) and biopsy (HR, 2.10; 95% CI, 1.43-3.07; P < .001) had shortened overall survival compared with GTR but no survival differences between them (HR, 0.91; 95% CI, 0.67-1.24; P = .56). Gross total resection was associated with prolonged survival compared with STR for hemispheric (HR, 0.29; 95% CI, 0.15-0.54; P < .001) and infratentorial (HR, 0.44; 95% CI, 0.24-0.83; P = .01) tumors but not midline tumors (HR, 0.63; 95% CI, 0.34-1.19; P = .16). Conclusions and Relevance: Results of this study show that, among patients with pHGG, GTR is independently associated with better overall survival compared with STR and biopsy, especially among patients with hemispheric and infratentorial tumors, and support the pursuit of maximal safe resection in the treatment of pHGGs.

Age-related injury responses of human oligodendrocytes to metabolic insults: link to BCL-2 and autophagy pathways.

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 (

Invasive growth associated with cold-inducible RNA-binding protein expression drives recurrence of surgically resected brain metastases.

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 >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.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles.

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.

Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

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.

Author Correction: Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.