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Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Diffuse midline gliomas driven by lysine27-to-methionine mutations in histone 3 (H3-K27M DMGs) are among the most fatal brain tumors. Molecular studies including single cell RNA-sequencing (scRNA-seq) of pediatric and predominantly pontine H3-K27M DMGs have shown that the H3-K27M oncohistone keeps glioma cells locked in a stem-like oligodendrocyte precursor cell (OPC) state that is capable of self-renewal and tumor-initiation. However, a comprehensive dissection of the cellular architecture of H3-K27M DMGs across different midline regions and age groups is required to better understand the cell-intrinsic and contextual regulation of H3-K27M DMG cell identities. In particular, the more recently described group of adult H3-K27M DMGs remains understudied. Here, we have collected and characterized 45 H3-K27M mutant patient tumors, spanning pontine (n=26), thalamic (n=17), and spinal (n=2) locations. Median age at surgery was 12 (2-68) years, encompassing 21 early childhood (0-10 years), 12 adolescent (11-20 years), and 12 adult (≥ 21 years) tumors. The majority of samples were obtained pre-treatment (n=28), as opposed to post-treatment or at autopsy (n=17). We profiled all 45 tumors by single cell/single nucleus RNA-seq and selected tumors were further characterized by the single cell assay for transposase-accessible chromatin (scATAC-seq). Our integrated analyses highlight the predominance of transcriptionally and epigenetically defined OPC-like tumor cells as the main cell population of H3-K27M DMGs across all age groups and locations. We further identify distinct age- and location-specific OPC-like cell subpopulations. Comparison of pediatric and adult tumors further demonstrates a significant increase of mesenchymal cell states in adult H3-K27M DMGs, which we link to differences in glioma-associated immune cell compartments between age groups. Together, this study sheds light on the effects of age- and region-dependent microenvironments in shaping cellular identities in H3-K27M DMGs.

Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/single nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets such as IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Medulloblastoma is a malignant childhood cerebellar tumor comprised of distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. We used single-cell transcriptomics to investigate intra- and inter-tumoral heterogeneity in twenty-five medulloblastomas spanning all molecular subgroups. WNT, SHH, and Group 3 tumors comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumors were exclusively comprised of differentiated neuronal-like neoplastic cells. SHH tumors closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumors exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, whose relative proportions distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide novel insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers. Single-cell analyses of glioblastoma samples reveal multiple cellular states, their plasticity and the genetic underpinnings of state proportions in a given tumor., National Cancer Institute (U.S.) (Grants 1U24CA180922, R33CA202820, P30CA14051)

High-grade gliomas harboring H3 G34R/V mutations exclusively occur in the cerebral hemispheres of adolescents and young adults, suggesting a distinct neurodevelopmental origin. Combining multimodal bulk and single-cell genomics with unbiased genome-scale CRISPR/Cas9 approaches, we here describe a GABAergic interneuron progenitor lineage as the most likely context from which these H3 G34R/V mutations drive gliomagenesis, conferring unique and tumor-selective gene targets essential for glioma cell survival, as validated genetically and pharmacologically. Phenotypically, we demonstrate that while H3 G34R/V glioma cells harbor the neurotransmitter GABA, they are developmentally stalled, and do not induce the neuronal hyperexcitability described in other glioma subtypes. These findings offer a striking counter-example to the prevailing view of glioma origins in glial precursor cells, resulting in distinct cellular, microenvironmental, and therapeutic consequences.

The work by Tang et al. provides a comprehensive, single-cell, transcriptomic analysis of kidney and blood cells from the adult zebrafish, identifying novel cell types, including two classes of NK immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells., Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.

Summary Medulloblastoma is a malignant childhood cerebellar tumour comprised of distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. We used single-cell transcriptomics to investigate intra- and inter-tumoural heterogeneity in twenty-five medulloblastomas spanning all molecular subgroups. WNT, SHH, and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours were exclusively comprised of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, whose relative proportions distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide novel insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

The apolipoprotein E (APOE) e4 allele is the major genetic risk factor for Alzheimer’s disease (AD). Multiple regulatory elements, spanning the extended TOMM40-APOE-APOC2 region, regulate gene expression at this locus. Regulatory element DNA methylation changes occur under different environmental conditions, such as disease. Our group and others have described an APOE CpG island as hypomethylated in AD, compared to cognitively normal controls. However, little is known about methylation of the larger TOMM40-APOE-APOC2 region. The hypothesis of this investigation was that regulatory element methylation levels of the larger TOMM40-APOE-APOC2 region are associated with AD. The aim was to determine whether DNA methylation of the TOMM40-APOE-APOC2 region differs in AD compared to cognitively normal controls in post-mortem brain and peripheral blood. DNA was extracted from human brain (n = 12) and peripheral blood (n = 67). A methylation array was used for this analysis. Percent methylation within the TOMM40-APOE-APOC2 region was evaluated for differences according to tissue type, disease state, AD-related biomarkers, and gene expression. Results from this exploratory analysis suggest that regulatory element methylation levels within the larger TOMM40-APOE-APOC2 gene region correlate with AD-related biomarkers and TOMM40 or APOE gene expression in AD.

Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME).We dissected these influences in isocitrate dehydrogenase (IDH)-mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses., National Cancer Institute (U.S.) (Grant P30-CA14051)

Summary Ependymoma is a heterogeneous entity of central nervous system tumors with well-established molecular groups. Here, we apply single-cell RNA sequencing to analyze ependymomas across molecular groups and anatomic locations to investigate their intratumoral heterogeneity and developmental origins. Ependymomas are composed of a cellular hierarchy initiating from undifferentiated populations, which undergo impaired differentiation toward three lineages of neuronal-glial fate specification. While prognostically favorable groups of ependymoma predominantly harbor differentiated cells, aggressive groups are enriched for undifferentiated cell populations. The delineated transcriptomic signatures correlate with patient survival and define molecular dependencies for targeted treatment approaches. Taken together, our analyses reveal a developmental hierarchy underlying ependymomas relevant to biological and clinical behavior., Graphical Abstract, Highlights • Cellular hierarchies in ependymoma reflect impaired neurodevelopment • Ependymoma cells follow neuronal-, astrocytic-, and ependymal-like trajectories • Undifferentiated programs confer inferior prognosis and are enriched at recurrence • Preclinical data suggest feasibility of inhibiting undifferentiated subpopulations, Gojo et al. use single-cell RNA sequencing to investigate intratumoral heterogeneity and cellular hierarchy in pediatric ependymoma, identifying impaired neurodevelopmental trajectories. The malignant trajectories reveal therapeutic targets and prognostic signatures in ependymoma.