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Joint profiling of chromatin accessibility and gene expression in individual cells provides an opportunity to decipher enhancer-driven gene regulatory networks (GRNs). Here we present a method for the inference of enhancer-driven GRNs, called SCENIC+. SCENIC+ predicts genomic enhancers along with candidate upstream transcription factors (TFs) and links these enhancers to candidate target genes. To improve both recall and precision of TF identification, we curated and clustered a motif collection with more than 30,000 motifs. We benchmarked SCENIC+ on diverse datasets from different species, including human peripheral blood mononuclear cells, ENCODE cell lines, melanoma cell states and Drosophila retinal development. Next, we exploit SCENIC+ predictions to study conserved TFs, enhancers and GRNs between human and mouse cell types in the cerebral cortex. Finally, we use SCENIC+ to study the dynamics of gene regulation along differentiation trajectories and the effect of TF perturbations on cell state. SCENIC+ is available at scenicplus.readthedocs.io . SCENIC+ is a comprehensive toolbox for inferring and analyzing enhancer-driven gene regulatory networks using single-cell multiomic data.

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment. Cancer stem cells (CSCs) comprise a putative population that can drive growth and resistance. Here, in glioblastoma models the authors show that rather than being a distinct clonal entity, the CSC population represents a plastic state adoptable by most cancer cells via reversible state transitions induced by the microenvironment.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications. Mechanical forces, along with gene regulatory networks and cell-cell signalling, play an important role in the complex organization of tissues. Here the authors describe devices that actively apply mechanical force to developing neural tube, demonstrating that mechanical forces increase growth and enhance patterning.

Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable ‘mixed’ GRN rather than being a symbiotic heterogeneous mix of cells.Profiling phenotype switching in melanoma. Wouters et al. perform large-scale gene expression analysis in patient samples and identify transcriptional networks that modulate the intermediate cell state and ultimate mesenchymal switching in melanoma.

In the mammalian liver, hepatocytes exhibit diverse metabolic and functional profiles based on their location within the liver lobule. However, it is unclear whether this spatial variation, called zonation, is governed by a well-defined gene regulatory code. Here, using a combination of single-cell multiomics, spatial omics, massively parallel reporter assays and deep learning, we mapped enhancer-gene regulatory networks across mouse liver cell types. We found that zonation affects gene expression and chromatin accessibility in hepatocytes, among other cell types. These states are driven by the repressors TCF7L1 and TBX3, alongside other core hepatocyte transcription factors, such as HNF4A, CEBPA, FOXA1 and ONECUT1. To examine the architecture of the enhancers driving these cell states, we trained a hierarchical deep learning model called DeepLiver. Our study provides a multimodal understanding of the regulatory code underlying hepatocyte identity and their zonation state that can be used to engineer enhancers with specific activity levels and zonation patterns. Bravo González-Blas et al. uncover enhancer-gene regulatory networks underlying hepatocyte identity and their zonation state by combining single-cell and spatial multiomics with massively parallel reporter assays and deep learning.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The brain cell types that control their sophisticated behavioral repertoire are still unknown. Here, we profile the cell diversity of the paralarval Octopus vulgaris brain to build a cell type atlas that comprises mostly neural cells, but also multiple glial subtypes, endothelial cells and fibroblasts. We spatially map cell types to the vertical, subesophageal and optic lobes. Investigation of cell type conservation reveals a shared gene signature between glial cells of mouse, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila . We construct a cell type taxonomy revealing transcriptionally related cell types, which tend to appear in the same brain region. Together, our data sheds light on cell type diversity and evolution in the octopus brain. The brain cell types of Octopus vulgaris that control their sophisticated behavioral repertoire are still unknown. Here, authors use single-cell transcriptomics to profile neuronal and glial cell types and compare cell type relationships within the octopus brain and across species.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson’s Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase ( TH ) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2 ). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1 , and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1 , ATRX , TP53 , MDM2/4 , amplification of EGFR , PDGFRA , MET , CDK4/6 , MDM2/4 , and deletion of CDKN2A/B , PTCH , and PTEN . Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Background A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. Methods Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. Results We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood–brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. Conclusions Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment. Graphical Abstract

Background Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson’s disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7 /DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. Methods Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7 /DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7 /DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). Results By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7 /DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7 /DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7 /DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7 /DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 h after acute inflammation, as also observed in BMDMs. Conclusions Taken together, our results show that, under inflammatory conditions, PARK7 /DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7 /DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.