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Neural–tumor interactions drive glioma growth as evidenced in preclinical models,but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients’ survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma,primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like,astrocyte-like and oligodendrocyte-progenitor-like,alongside oligodendrocytes and excitatory neurons. In line with these findings,high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients,a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients’ plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes. Subject terms: Translational research,CNS cancer,DNA methylation View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans,respectively. These vasoformative sarcomas are aggressive and highly metastatic,with disorganized,irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized,and they were lined by both donor and host cells. In a series of xenografts,we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore,gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas,and possibly human angiosarcomas,maintain molecular properties that provide hematopoietic support and facilitate stromal reactions,suggesting their potential involvement in promoting the growth of hematopoietic tumors. We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation,providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression. View Publication

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation,but whether and how metabolic sensor O -GlcNAcylation,which can be modulated via an inhibition of its cycling enzymes O -GlcNAcase (OGA) and O -GlcNAc transferase (OGT),contributes to hematopoiesis remains largely unknown. Herein,isogenic,single-cell clones of OGA -depleted (OGAi) and OGT -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A,which were reprogrammed from CD34 + hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O -GlcNAcylation concomitant with their loss of OGA and OGT,respectively,appeared normal in phenotype and karyotype,and retained pluripotency,although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition,we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors,thus confirming their functional characteristics. Altogether,the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation,i.e.,by small molecules,allowing the molecular dynamics studies of O -GlcNAcylation. View Publication

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem‐like cancer cells,called glioblastoma stem cells (GSCs). GSCs have the capacity for self‐renewal and tumorigenesis,leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem‐like cancer cells,their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation,novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image‐based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs,facilitating the development of NK cell‐based immunotherapy for improved GBM treatment. View Publication

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however,an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4 + MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity,but MP cells retain less compromised mitochondria compared to effector CD4 + T cells,and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells. Subject areas: immunity,cell biology View Publication

Solid cancers Myeloid cells are prevalent in solid cancers,but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME),which hinders the effectiveness of cancer immunotherapies. Myeloid cells’ natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers,including tumor infiltration,tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1,which is often upregulated by solid cancers to evade immune responses. Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1,while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells,we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally,we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy’s effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1β. Moreover,CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors,infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together,these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers. View Publication

Early childhood tumours arise from transformed embryonic cells,which often carry large copy number alterations (CNA). However,it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains,which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives,the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN,whose amplification co-occurs with CNAs in NB. Moreover,CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together,our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours. Subject terms: Paediatric cancer,Stem cells,Disease model,Cancer genomics,Embryonal neoplasms View Publication

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC),which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover,immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids,which will advance the use of kidney organoids for transplantation research. View Publication

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines. View Publication

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

Allogeneic cell therapies,such as those involving macrophages or Natural Killer (NK) cells,are of increasing interest for cancer immunotherapy. However,the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges,such as required cell pre-activation and inefficiency in transduction,which hinder the assessment of preclinical efficacy and clinical translation. In our study,we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein,which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors,this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood,as well as freshly obtained monocytes,which were differentiated to M1 macrophages as well as B cells from multiple donors,achieving up to 80% reporter gene expression within three days post-transduction. Importantly,KoRV-based transduction does not compromise the expression of crucial immune cell receptors,nor does it impair immune cell functionality,including NK cell viability,proliferation,cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion,our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics,requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics,expediting their availability to patients in need. Subject terms: Genetic transduction,Tumour immunology,Immunotherapy,Genetic vectors,Innate immune cells View Publication