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Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by extensive intratumoral heterogeneity. To investigate the underlying biology, we conducted single-cell RNA-sequencing (scRNA-seq) of >1500 cells from six primary TNBC. Here, we show that intercellular heterogeneity of gene expression programs within each tumor is variable and largely correlates with clonality of inferred genomic copy number changes, suggesting that genotype drives the gene expression phenotype of individual subpopulations. Clustering of gene expression profiles identified distinct subgroups of malignant cells shared by multiple tumors, including a single subpopulation associated with multiple signatures of treatment resistance and metastasis, and characterized functionally by activation of glycosphingolipid metabolism and associated innate immunity pathways. A novel signature defining this subpopulation predicts long-term outcomes for TNBC patients in a large cohort. Collectively, this analysis reveals the functional heterogeneity and its association with genomic evolution in TNBC, and uncovers unanticipated biological principles dictating poor outcomes in this disease. Triple-negative breast cancer is highly heterogeneous and aggressive. Here, the authors utilise single-cell RNA sequencing to investigate this heterogeneity, and discover a subpopulation of cells associated with metastasis and treatment resistance signatures, and linked to long term survival outcomes.

Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.

Methods for quantifying gene expression 1 and chromatin accessibility 2 in single cells are well established, but single-cell analysis of chromatin regions with specific histone modifications has been technically challenging. In this study, we adapted the CUT&Tag method 3 to scalable nanowell and droplet-based single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues and during the differentiation of human embryonic stem cells. We focused on profiling polycomb group (PcG) silenced regions marked by histone H3 Lys27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we used scCUT&Tag to profile H3K27me3 in a patient with a brain tumor before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment. An improved method for single-cell analysis of histone modifications is applied to stem cell differentiation and cancer.

The accepted paradigm for both cellular and anti-tumor immunity relies upon tumor cell killing by CD8 + T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex (MHC) class I (MHC-I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8 + T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell natural killer group 2D (NKG2D) and tumor NKG2D ligands (NKG2DLs), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell killing in these instances is antigen independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice as well as in vitro in human tumor systems and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape and instead identify the NKG2D–NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC-loss variants.

Quiescence cancer stem-like cells may play key roles in promoting tumor cell heterogeneity and recurrence for many tumors, including glioblastoma (GBM). Here we show that the protein acetyltransferase KAT5 is a key regulator of transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states in GBM. KAT5 activity suppresses the emergence of quiescent subpopulations with neurodevelopmental progenitor characteristics, while promoting GBM stem-like cell (GSC) self-renewal through coordinately regulating E2F- and MYC- transcriptional networks with protein translation. KAT5 inactivation significantly decreases tumor progression and invasive behavior while increasing survival after standard of care. Further, increasing MYC expression in human neural stem cells stimulates KAT5 activity and protein translation, as well as confers sensitivity to homoharringtonine, to similar levels to those found in GSCs and high-grade gliomas. These results suggest that the dynamic behavior of KAT5 plays key roles in G0 ingress/egress, adoption of quasi-neurodevelopmental states, and aggressive tumor growth in gliomas. Quiescent populations are a likely key source of treatment resistance in glioblastoma. Here, the authors characterize KAT5 as a critical mediator of quiescence and adoption of progenitor-like states.

Glioblastoma (GBM) remains an untreatable malignant tumor with poor patient outcomes, characterized by palisading necrosis and microvascular proliferation. While single-cell technology made it possible to characterize different lineage of glioma cells into neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal like (MES-like) states, it does not capture the spatial localization of these tumor cell states. Spatial transcriptomics empowers the study of the spatial organization of different cell types and tumor cell states and allows for the selection of regions of interest to investigate region-specific and cell-type-specific pathways. Here, we obtained paired 10x Chromium single-nuclei RNA-sequencing (snRNA-seq) and 10x Visium spatial transcriptomics data from three GBM patients to interrogate the GBM microenvironment. Integration of the snRNA-seq and spatial transcriptomics data reveals patterns of segregation of tumor cell states. For instance, OPC-like tumor and NPC-like tumor significantly segregate in two of the three samples. Our differentially expressed gene and pathway analyses uncovered significant pathways in functionally relevant niches. Specifically, perinecrotic regions were more immunosuppressive than the endogenous GBM microenvironment, and perivascular regions were more pro-inflammatory. Our gradient analysis suggests that OPC-like tumor cells tend to reside in areas closer to the tumor vasculature compared to tumor necrosis, which may reflect increased oxygen requirements for OPC-like cells. In summary, we characterized the localization of cell types and tumor cell states, the gene expression patterns, and pathways in different niches within the GBM microenvironment. Our results provide further evidence of the segregation of tumor cell states and highlight the immunosuppressive nature of the necrotic and perinecrotic niches in GBM.

Abstract BACKGROUND: Glioblastoma is a fatal brain tumor in needing urgent effective therapy. Treatments with both oncolytic viruses and immunotherapy have shown preclinical efficacy and clinical promise. We sought to exploit possible synergies between oncolytic herpes simplex virus type 1 (oHSV-1) infection of intracranial gliomas and delivery of immune-stimulating fms-like tyrosine kinase 3 ligand (Flt3L) by engineering a herpes vector to express the cytokine. OBJECTIVE: To construct an oHSV-1 vector that expresses high levels of Flt3L and examine its antiglioma efficacy in an immunocompetent murine model. METHODS: G47Δ and a bacterial artificial chromosome system were used to generate a novel oHSV-1, termed G47Δ-Flt3L, expressing Flt3L. Cytokine expression was confirmed, and G47Δ-Flt3L was injected intratumorally into established intracranial CT-2A gliomas in syngeneic C57/Bl6 mice. Animals were followed for survival and assessed by the Kaplan-Meier method. RESULTS: G47Δ-Flt3L expressed high levels of Flt3L in culture. Expression of Flt3L affected neither viral replication nor had a cytotoxic effect on CT2A glioma cells. Direct inoculation into intracerebral CT2A glioma cells resulted in high levels of detectable Flt3L in mouse blood and was superior to parental G47Δ in prolonging survival in glioma-bearing animals. CONCLUSION: Treatment with G47Δ-Flt3L improves survival of glioma-bearing mice.

Glioblastoma (GBM) is a heterogeneous tumor made up of cell states that evolve over time. Here, we modeled tumor evolutionary trajectories during standard-of-care treatment using multi-omic single-cell analysis of a primary tumor sample, corresponding mouse xenografts subjected to standard of care therapy, and recurrent tumor at autopsy. We mined the multi-omic data with single-cell SYstems Genetics Network AnaLysis (scSYGNAL) to identify a network of 52 regulators that mediate treatment-induced shifts in xenograft tumor-cell states that were also reflected in recurrence. By integrating scSYGNAL-derived regulatory network information with transcription factor accessibility deviations derived from single-cell ATAC-seq data, we developed consensus networks that modulate cell state transitions across subpopulations of primary and recurrent tumor cells. Finally, by matching targeted therapies to active regulatory networks underlying tumor evolutionary trajectories, we provide a framework for applying single-cell-based precision medicine approaches to an individual patient in a concurrent, adjuvant, or recurrent setting.

OBJECTIVES/GOALS: Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. METHODS/STUDY POPULATION: Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. RESULTS/ANTICIPATED RESULTS: In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. DISCUSSION/SIGNIFICANCE: Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

A breakthrough in drug discovery for glioblastoma requires serial collection of tissue from the central nervous system via window of opportunity trials