Search PubMedSearch

SEARCH · Search PubMed

Results for “ScRNA-seq”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

Localized PD-1 CAR T therapy reprograms neuroinflammation.

B cell-depleting therapies are effective in multiple sclerosis (MS), yet some patients relapse, underscoring the need for more precise interventions. To identify new therapeutic targets, we generated a single-cell RNA sequencing (scRNA-seq) atlas of cerebrospinal fluid (CSF), brain, and blood from non-inflammatory controls and patients with MS or other neuroinflammatory diseases. We found disease-associated enrichment of class-switched immunoglobulin G+ (IgG+) B cells and plasma cells in MS CSF. Unbiased analysis identified a rare disease-enriched subset of activated, T cell receptor (TCR)-restricted, PD-1+ T follicular helper-like cells with B cell-recruiting features. To target this population, we developed PD-1-directed chimeric antigen receptor (CAR) T cells that selectively depleted pathogenic PD-1+ CD4 T cells and locally released IL-10. This strategy attenuated central nervous system (CNS) inflammation, reprogrammed the local immune milieu, and improved clinical outcomes across murine neuroinflammation models. These findings define a CNS-localized adaptive immune circuit in MS and nominate programmable PD-1 CAR T cells as a strategy to disrupt it.

Animals

Single-cell transcriptional profiling identifies the swimming crab Portunus trituberculatus in response to bacterial infection.

Crustaceans rely entirely on innate immunity, yet the cellular composition, functional specialization, and pathogen-induced remodeling of their immune system remain poorly resolved. Here, we generated a high-resolution single-cell transcriptomic atlas of hemocytes from the swimming crab Portunus trituberculatus following Vibrio parahaemolyticus infection using 10× Genomics scRNA-seq. Seven putatively distinct hemocyte clusters were identified, including granulocytes, semigranular hemocytes, prohemocytes, unresolved hemocytes, hyalinocyte-like hemocytes, biosynthetically active secretory hemocytes, and regulatory hemocytes. Although the overall cellular composition remained relatively stable after infection, hemocytes exhibited pronounced cluster-specific transcriptional reprogramming involving Toll/NF-κB signaling, antimicrobial peptide synthesis and metabolic rewiring. By integrating single-cell and bulk transcriptomes, we identified multiple anti-lipopolysaccharide factors (ALFs) as key secretory effectors and experimentally validated their antibacterial activities. FITC-based bacterial engulfment assays and RNA-seq of sorted phagocytes demonstrated that phagocytic capability was shared across multiple hemocyte clusters. Notably, the immunoglobulin superfamily receptor DSCAM displayed extensive alternative splicing and strong infection-induced activation in unresolved hemocytes. Immune-training experiments showed that prior bacterial exposure was associated with altered DSCAM expression and reduced early cumulative mortality upon secondary challenge, suggesting a memory-like immune phenotype. These findings provide a foundational framework for understanding crustacean immunity and advancing disease-resistant breeding in aquaculture.

Antimicrobial peptides

Ligand-based directed differentiation to produce granulosa-like cells expressing steroidogenic enzyme genes.

The ovarian granulosa cells are responsible for producing hormones and supporting oocytes through maturation and meiotic resumption. There is a need to generate granulosa-like cells (GLCs) from human induced pluripotent stem cells (hiPSCs) to better model human gonadal development and to test the effects of exogenous or pharmaceutical compounds on the ovary. Here we report a rapid ligand-based protocol for differentiating hiPSCs into cells that express markers of the transient developmental lineages and steroidogenic pathway genes. Single-cell RNA-sequencing (scRNA-seq) analysis identified canonical granulosa cell genes were expressed in a subset of cells and identified new genes of interest that were significantly associated with computationally modeled pseudotime. HSD17B1 was expressed in resulting GLCs but at low levels, suggesting an immature granulosa cell phenotype. The GLCs were produced using a simple culture method that could be augmented for granulosa cell functions such as sustaining oocyte growth. Producing GLCs through protocols such as this one is a first step toward designing large-scale ovarian endocrinology assays and developing personalized cell-based fertility and hormone restoration technologies in the future. This rapid protocol produced cells that express steroidogenic enzyme genes etoc blurb. Kubo and colleagues present a 5-day rapid protocol to generate immature granulosa-like cells from hiPSCs. Cells differentiated with inhibition of DKK1, a WNT signaling target gene, expressed gonadal ridge markers and FOXL2 transcripts and protein. Additionally, steroidogenic enzyme genes were expressed. A small population of differentiated cells were identified as expressing early-stage granulosa cell genes by single-cell RNA-seq.

Female

Quantitative molecular cartography of emergency myelopoiesis reveals conserved modules of hematopoietic activation.

Hematopoietic stem and progenitor cells (HSPCs) respond to infections, inflammation, and regenerative challenges using emergency myelopoiesis (EM) pathways to amplify myeloid cell production. However, it remains unclear how various EM inducers regulate HSPCs using shared or distinct molecular mechanisms. Here, we generate a comprehensive and generalizable cell annotation method (HemaScribe) and a refined quantitative model of hematopoietic differentiation (HemaScape) using single-cell RNA sequencing (scRNA-seq) of murine HSPCs, which we apply to a broad range of EM modalities. We uncover multiple strategies for enhancing myelopoiesis that act at different levels of the HSPC hierarchy and are associated with both unique and shared transcriptional response modules. In particular, we identify a myeloid progenitor-based EM activation module across diverse inflammatory challenges that is conserved in humans and informs outcomes in adult and pediatric acute myeloid leukemia. Our work illuminates fundamental regulatory mechanisms in hematopoietic regeneration that have direct translational applications in disease contexts.

Animals

Dynamic evolution of chaperone-mediated autophagy is associated with tumor microenvironment remodeling and prognostic stratification in lung adenocarcinoma: insights from single-cell transcriptomics, ensemble machine learning, and experimental validation.

BACKGROUND: Lung adenocarcinoma (LUAD) shows prognostic heterogeneity, and tumor-node-metastasis (TNM) staging is limited for individualized management. Chaperone-mediated autophagy (CMA) maintains proteostasis, but its role during adenocarcinoma in situ (AIS)-minimally invasive adenocarcinoma (MIA)-invasive adenocarcinoma (IAC) progression remains unclear. METHODS: Single-cell RNA sequencing (scRNA-seq) data from GSE189357 and bulk transcriptomes from The Cancer Genome Atlas (TCGA)-LUAD and Gene Expression Omnibus (GEO) cohorts were integrated. CMA activity, cell-cell communication, weighted gene co-expression network analysis (WGCNA), tumor-normal differential expression, machine-learning survival modeling, tumor microenvironment (TME) features, drug sensitivity, and EPC1 function were analyzed. RESULTS: CMA-high tumor epithelial cells increased from AIS (58.1%) to MIA (65.7%) but declined in IAC (44.4%; p < 0.001). CMA-low cells preferentially received fibroblast-derived extracellular matrix cues. A CMA-negatively correlated module identified 69 core genes. Random survival forest (RSF) performed best among 117 machine-learning combinations (mean concordance index > 0.873). High-risk patients had worse survival across cohorts, and the risk score was independently associated with overall survival (hazard ratio = 16.013, 95% confidence interval: 9.579-26.768, p < 0.001). High-risk tumors showed proliferative activation and M0 macrophage enrichment, whereas low-risk tumors showed stronger immune-related signaling. EPC1 overexpression suppressed malignant phenotypes in A549 cells. CONCLUSION: CMA dynamics are associated with stromal and immune remodeling during LUAD progression. A CMA-based model provides robust prognostic stratification and may offer a basis for future TME-guided studies.

Chaperone-mediated autophagy

Integrated single-cell and bulk transcriptomic analysis identifies a novel senescent fibroblast subtype associated with poor prognosis in acral melanoma.

BACKGROUND: Acral melanoma (AM) exhibits significant intratumoral heterogeneity, but its tumor microenvironment (TME) and immune regulation remain unclear. This study aims to dissect TME heterogeneity and establish a prognostic model based on key cell subpopulations. METHODS: We collected AM single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA). Unsupervised clustering, CellChat, and Scissor analysis were performed to characterize cellular heterogeneity, cell-cell communication, and prognosis-related cell subpopulations. Kaplan-Meier analysis was used to assess the prognostic value of key genes, which were further validated by multiplex immunohistochemistry (mIHC). RESULTS: In AM, Mel_C2, C7, and C9 with high SEMA6A and KIT expression were strongly linked to poor prognosis. We further identified a senescent fibroblast subpopulation (sCAF_CDKN2A) characterized by high fibroblast senescence signature (FSS) scores. Integrating Scissor analysis of fibroblast subtypes with bulk prognostic data, we identified COL3A1, VCAN, and KIT as prognosis-associated genes upregulated in poor-outcome-related fibroblast subsets. Cell-cell communication analysis revealed that sCAF_CDKN2A engages in an immunosuppressive network, interacting with regulatory T cells (Tregs) via MIF signaling and receiving signals from exhausted CD8+ T cells through PPIA-BSG interactions. Using transcription factor expression patterns from these fibroblast subtypes, we constructed a prognostic model that effectively stratified patients into distinct risk groups with significant differences in overall survival (OS). mIHC confirmed significantly higher protein levels of SEMA6A and COL3A1 in tumor tissues compared to matched normal tissues. CONCLUSIONS: We established a novel prognostic model for AM and identified sCAF_CDKN2A as an immunosuppressive senescent fibroblast subpopulation driving poor prognosis.

Acral melanoma

Rbp-J&#x3ba; controls NK cell late maturation and migration via chromatin landscape remodeling.

The transcriptional regulator Rbp-J&#x3ba; is a pivotal molecular switch in Notch signaling; however, its cell-intrinsic role in natural killer (NK) cell maturation and migration remains incompletely understood. Here, we demonstrate that NK cell-specific deletion of Rbp-J&#x3ba; (Ncr1iCre &#xd7; Rbp-J&#x3ba;fl/fl, Rbp-J&#x3ba;&#x394;NK) impairs NK cell terminal maturation and migration, as evidenced by increased retention of NK cells in bone marrow, a reduced number of circulating NK cells and decreased expression of migration mediators (CD62L, S1pr5, and Cx3cr1). Despite exhibiting an activated phenotype, Rbp-J&#x3ba;-deficient NK cells fail to control B16F10 lung metastases in vivo because of impaired tissue mobilization. Multiomics (scRNA-seq/scATAC-seq, bulk ATAC-seq, and CUT&Tag) reveal that Rbp-J&#x3ba; orchestrates chromatin remodeling in NK cells, suppressing the expression of genes related to NK cell activation and cytotoxicity while promoting the expression of genes involved in ribosome and oxidative phosphorylation. Notably, Rbp-J&#x3ba; directly binds to the Kruppel-like factor 2 (Klf2) promoter, and loss of Rbp-J&#x3ba; reduces both the mRNA and protein levels of Klf2. Klf2 overexpression rescues the decreased expression of CD62L and CX3CR1 in Rbp-J&#x3ba;-deficient NK cells. The cooccupancy of Rbp-J&#x3ba; and Klf2 at shared genomic loci is confirmed by ChIP-qPCR. In summary, our study reveals that Rbp-J&#x3ba; acts as a master regulator of NK cell terminal maturation and tissue homing via chromatin reprogramming, with Klf2 acting as its critical downstream transcription factor.

Animals

Nested co-expression network analysis identifies compact gene clusters in a black box.

MOTIVATION: Digital analysis of biological systems requires methods capable of identifying both broad and nested gene modules reflecting complex biological processes. Existing transcriptomic methods often miss compact gene sets corresponding to subprocesses in specialized cell types, limiting insights into functional heterogeneity. RESULTS: We present Nested-WGCNA, a two-stage unsupervised network analysis algorithm designed to identify coarse-grained and fine-grained gene modules. Applied to bulk RNA-Seq data, Nested-WGCNA reveals stable modules reproducible across datasets. When validated against scRNA-Seq data, these modules correspond to both major and minor immune cell subtypes. Application to immunotherapy response datasets uncovers predictive and prognostic biomarkers, highlighting its utility in treatment stratification and biomarker discovery. AVAILABILITY: The NestedWGCNA source code and analysis pipeline are available on GitHub (https://github.com/ilyada/NestedWGCNA) and archived on Zenodo (https://doi.org/10.5281/zenodo.18959244).

Algorithms

Integrated multi-omics strategies for identifying novel therapies in psoriasis.

MOTIVATION: Psoriasis is a chronic, immune-mediated disorder with an unmet need for effective treatments. To systematically prioritize therapeutic targets, we integrated proteome-wide Mendelian randomization (MR) with expression validation in blood/skin, genetic susceptibility analysis, differential gene expression (DGE) from bulk and single-cell RNA sequencing (scRNA-seq), colocalization, pathway enrichment, and protein-protein interaction analyses. RESULTS: Proteome-wide MR identified 29 candidate protein targets (Bonferroni-corrected), all replicated in independent datasets. Fifteen targets showed significant expression associations in blood or skin. Eleven proteins-UBLCP1, IL23A, ASF1A, RARRES2, ICAM1, PRSS53, ICAM5, GCA, IL2RA, DBI, and NFKB1-exhibited consistent directional effects with their genes. Genetic susceptibility analysis confirmed 20 target-specific polygenic scores for psoriasis and five for psoriatic arthritis. DGE analysis identified 13 targets in bulk and 13 in scRNA-seq-primarily in keratinocytes and immune cells-with IL2RA, COMP, and A2ML1 dysregulated across both. Colocalization analysis implicated shared causal variants for psoriasis in ASF1A, CD8A, CTF1, IL7R, MMP12, RARRES2, XCL2, DBI, IL23A, IL2RA, SGSH, and TIMD4. Enrichment analyses highlighted involvement in cytotoxicity, immune regulation, and JAK-STAT signaling. Eighteen targets interacted with approved anti-psoriasis drugs. Notably, drugs targeting IL2RA, IL7R, CTF1, ICAM1, MMP12, NFKB1, CD8A, DDX58, IL12A, SGSH, and FAP are approved or in trials for other diseases, suggesting repurposing potential. Our integrative multi-omics approach prioritized 29 high-confidence targets, including 13 novel candidates (RARRES2, ASF1A, CTF1, DBI, B3GNT2, CD8A, TIMD4, CRTAM, SGSH, XCL2, DAPK2, A2ML1, and FAP). Several high-priority targets-such as IL2RA, IL23, MMP12, RARRES2, IL7R, and ICAM1-were supported across analytical layers. These findings provide a robust foundation for psoriasis drug development. AVAILABILITY AND IMPLEMENTATION: The code used for the analyses in this manuscript has been archived in Zenodo at [DOI: 10.5281/zenodo.19692128].

Psoriasis

FIERCE: reconstructing dynamic trajectories from the differentiation potency of single cells.

MOTIVATION: Since the introduction of single-cell RNA sequencing (scRNA-seq), numerous computational approaches have been developed to reconstruct dynamic cellular processes from static transcriptional profiles. These methods order cells along continuous trajectories by assessing their similarity in the gene-expression space. However, they rely on several assumptions, such as prior knowledge of the structure and directionality of the expected genealogy. These assumptions can limit their application to complex cellular systems with poorly understood developmental paths. RESULTS: To address this challenge, we introduce FIERCE (Framework for InfERence of the veloCity of Entropy), a novel computational pipeline designed to predict the changes in the differentiation potency of single cells during dynamic processes. Through a fully unsupervised approach, FIERCE enables the inference of cell lineages directly on the differentiation landscape of the biological system, thus eliminating the need for prior specification of developmental parameters. We demonstrate the efficacy of FIERCE by reconstructing three well-known mouse differentiation systems and by quantifying its accuracy on simulated data. AVAILABILITY AND IMPLEMENTATION: The FIERCE R package is available on GitHub at https://github.com/bicciatolab/FIERCE.

Cell Differentiation

TMEM176B is co-expressed with TMEM176A and upregulated in peripheral blood monocytes of patients with primary Sj&#xf6;gren's syndrome.

OBJECTIVES: This study aims to determine the role of acid&#x2011;sensitive nonspecific cation channels transmembrane protein 176A (TMEM176A) and TMEM176B in autoimmune diseases, with a focus on primary Sj&#xf6;gren's Syndrome (pSS). METHODS: We examined the expression patterns of TMEM176A and TMEM176B across tissues and cells utilizing bulk RNA-seq and scRNA-seq datasets. Immunophenotyping analysis was performed by flow cytometry to compare CD62L expression between TMEM176B&#x207a; and TMEM176B&#x207b; monocytes. The proportion of TMEM176B+ cells in monocytes was interrogated in both pSS patients and healthy controls. Clinical correlations of TMEM176B with anti&#x2011;SSB antibody and complement C4 levels were also evaluated. RESULTS: TMEM176A and TMEM176B showed conserved co&#x2011;expression and were significantly upregulated in autoimmune diseases. TMEM176B&#x207a; monocytes displayed higher CD62L positivity rate than TMEM176B- monocytes. In pSS patients, the proportion of TMEM176B&#x207a; monocytes was elevated in total monocytes, classical monocytes (cMo) and intermediate monocytes (iMo). The proportion of TMEM176B+ monocytes positively correlated with anti&#x2011;SSB levels, while several TMEM176B-associated monocyte subset markers inversely correlated with C4. CONCLUSION: TMEM176A and TMEM176B are highly correlated. TMEM176B expression in monocytes is linked to pSS and may serve as a novel auxiliary diagnostic biomarker.

Humans

Spatial proteomic mapping of the human and mouse retina using IBEX.

We generated a comparative spatial proteomic atlas of the human and mouse retina using a highly multiplexed immunohistochemistry technique called iterative bleaching extends multiplexity (IBEX). We refined the IBEX workflow by integrating an antibody dissociation option alongside chemical bleaching. This dual strategy enabled removal of the entire antibody complex, permitting the flexible use of antibodies from the same host species across iterative cycles. We coupled this workflow with super-resolution imaging via deconvolution and applied it to the retina of healthy humans and WT mice and the Crb1rd8 mouse model. We successfully imaged over 25 protein markers on human and mouse tissue sections, generating spatial atlases of the major retinal cell populations. Cross-species protein expression was compared to scRNA-seq datasets to identify protein and transcript disparities. Super-resolution IBEX delineated the ultrastructural features of the outer limiting membrane (OLM), identifying CD44 as a core structural component tightly colocalized with a highly organized F-actin belt within M&#xfc;ller glial endfeet. Using the Crb1rd8 mouse model, disruption of this complex was spatially associated with rosette formation and OLM structural failure. In summary, spatial proteomic atlases of the human and mouse retina were used to reveal insights into the arrangement of major retinal cell populations and OLM structure.

Animals

Epigenetic and immunological alterations in umbilical cord blood of overweight/obese women with gestational diabetes mellitus: insights into DNA methylation signatures and immune cell dysregulation.

BACKGROUND: Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with adverse maternal and neonatal outcomes. Epigenetic modifications may reflect intrauterine metabolic exposure and contribute to immune and metabolic alterations. This study aimed to explore DNA methylation profiles in umbilical cord blood from overweight and obese women with and without GDM. METHODS: Umbilical cord blood samples from 30 overweight/obese pregnant women (with and without GDM) were analyzed using the Illumina 850&#xa0;K methylation array to identify differentially methylated positions (DMPs) and regions (DMRs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to assess the functional relevance of methylation changes. Immune cell composition was estimated using deconvolution analysis and further examined in an independent single-cell RNA sequencing (scRNA-seq) cohort. Lasso regression was applied to identify CpG sites associated with GDM status and construct a preliminary methylation-based classification model. RESULTS: A total of 23,331 hypermethylated and 29,501 hypomethylated DMPs were identified between women with and without GDM, with hypomethylation predominating. Enrichment analyses indicated associations with neurodevelopmental pathways, metabolic processes, immune regulation, and epigenetic modification. Immune deconvolution analysis suggested reduced proportions of CD4+ T cells (p&#x2009;<&#x2009;0.05) and a trend toward decreased NK cells in the GDM group, alongside increased CD8+ T cells and neutrophils. Seven CpG sites were selected for model construction and demonstrated strong discriminatory performance within this cohort. CONCLUSION: This exploratory study identifies distinct cord blood DNA methylation patterns associated with GDM in overweight/obese pregnancies. The findings suggest potential links between epigenetic alterations and immune cell composition in GDM-exposed offspring. The identified CpG signature warrants further validation in larger, prospective cohorts to determine its clinical applicability.

Humans

Hepatic metabolic adaptation to endurance exercise: temporal and sex differences by multiomics integration and validation.

BACKGROUND: Although endurance exercise benefits liver health, sex-specific adaptive trajectories remain unclear. This study mapped dynamic liver adaptation in males and females during prolonged training and identified underlying molecular programs. METHODS: Using publicly available time-resolved liver multi-omics data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we established a computational pipeline for differential analysis of transcriptomic, proteomic, phosphoproteomic, and metabolomic data with FDR correction, followed by FGSEA pathway enrichment. Kinase activities were inferred through ortholog mapping and PhosphoSitePlus. Cross-omics co-expression networks were constructed using WGCNA and topological overlap to link omics features with physiological phenotypes. For experimental validation, liver tissues were collected from endurance-trained Sprague-Dawley rats, and key nodes were confirmed by Western blotting, qRT-PCR, and immunofluorescence/immunohistochemical staining. Public scRNA-seq data were further integrated to map multi-omics signals to single-cell resolution and assess functional changes in specific cell types. RESULTS: The hepatic response to exercise stress was stage-specific, shifting from early transcriptional activation to later proteomic and metabolic remodeling. Multi-omics integration revealed distinct sex-associated adaptive trajectories: males were more strongly associated with energy metabolism, redox-related programs, and amino acid/organic acid catabolism, whereas females showed prominent membrane lipid remodeling, proteostasis -related programs, and mitochondrial/ribosomal translational features. Single-cell analysis showed that tissue remodeling occurred without major lineage turnover, instead involving altered communication among pre-existing cell communities. Validation of PPP1R3G identified a protein-dominant exercise-responsive marker, supporting the contribution of post-transcriptional or protein-level regulation. CONCLUSIONS: Hepatic adaptation to endurance stress follows a cross-omics evolutionary pattern with sex-specific reprogramming of energy supply and homeostatic maintenance. This time-resolved framework clarifies how exercise improves liver function and supports sex-oriented metabolic interventions and therapeutic target discovery.

Animals

MX1+ effector T cells hyperactivation at the maternal-fetal interface in unexplained recurrent pregnancy loss.

BACKGROUND: Immune tolerance breakdown at the maternal-fetal interface is implicated in unexplained recurrent pregnancy loss (URPL), but the interplay between T cell hyperactivation and dendritic cells (DCs)-mediated signaling remains poorly defined. METHODS: First-trimester decidual tissues from 5 healthy controls and 6 URPL patients underwent single-cell RNA sequencing (scRNA-seq, 10&#xd7; Genomics). Computational analyses included clustering (Seurat), trajectory inference (scTour), intercellular communication (CellChat) and metabolic pathway enrichment (Gene Ontology and scMetabolism). Flow cytometry was performed from 11 patients and 11 healthy controls. Spatial validation was performed via multiplex immunohistochemistry and immunohistochemistry on 12 additional controls and 12 URPL cases. Statistical significance was assessed using Student&#x2019;s t-test. RESULTS: URPL decidua exhibited marked CD3+ T cells and MX1+effector T (Tem) cells infiltration and activation. Flow cytometry analysis confirmed a significant decidua-specific upregulation of T cell activation markers CD25 and CD69 specifically on the MX1+Tem subset in URPL patients compared to controls. MX1+Tem cell subset demonstrated interferon hyperactivation, proliferative hyperactivity and lipid-biased immunometabolism. Pseudotemporal analysis positioned MX1+ Tem cells between classical Tem and exhausted T cell states, suggesting progressive differentiation. CellChat identified DCs as key regulators of MX1+ Tem expansion via aberrant ICOSL signaling, validated by spatial co-localization of ICOSL+ DCs and MX1+ Tem cells in URPL tissues. CONCLUSION: Our findings demonstrate that the aberrant activation and proliferation of MX1+Tem cells as a key immunological feature associated with URPL patients.

Humans

Non-structural maintenance of chromosome condensin I complex subunit H knockdown suppresses malignant progression of esophageal squamous cell carcinoma via the Wnt/&#x3b2;-catenin signaling pathway.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) remains a major cause of cancer-related mortality, and effective therapeutic targets are still limited. Non-structural maintenance of chromosome condensin I complex subunit H (NCAPH) has been implicated in tumorigenesis; however, its clinical relevance, functional roles, and underlying mechanisms in ESCC are not fully defined. We aimed to characterize the expression pattern, prognostic value, biological functions, and mechanistic basis of NCAPH in ESCC. METHODS: Public datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed to evaluate NCAPH expression and clinical associations. Single-cell RNA sequencing (scRNA-seq) data were used to map cell-type-specific distribution of NCAPH in tumor and adjacent tissues. NCAPH was silenced in KYSE150 and KYSE510 cells using lentiviral short hairpin RNAs (shRNAs), followed by Cell Counting Kit-8 (CCK-8), colony formation, wound-healing, and Transwell migration/invasion assays. A nude mouse xenograft model was established to assess the effect of NCAPH knockdown in vivo. RNA sequencing (RNA-seq), quantitative polymerase chain reaction (qPCR), western blotting, and enzyme-linked immunosorbent assay (ELISA) were performed to explore potential mechanisms. RESULTS: NCAPH was consistently upregulated in ESCC across multiple cohorts and was associated with unfavorable clinicopathological features and poorer survival. Functional assays demonstrated that NCAPH knockdown significantly inhibited ESCC cell proliferation, migration, invasion, and clonogenic growth. In vivo, NCAPH silencing suppressed xenograft tumor growth. Mechanistically, transcriptomic profiling and molecular validation indicated attenuation of Wnt/&#x3b2;-catenin signaling following NCAPH depletion, accompanied by reduced &#x3b2;-catenin and downstream targets. CONCLUSIONS: NCAPH promotes malignant progression of ESCC, at least in part through activation of the Wnt/&#x3b2;-catenin pathway, and may serve as a potential biomarker and therapeutic target.

Esophageal squamous cell carcinoma (ESCC)

Construction of molecular signatures based on the co-expression network of NECSO-related gene TRPM4 and its prognostic value in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) demonstrates significant prognostic variability that is not entirely accounted for by traditional staging systems. Necrosis by sodium overload (NECSO) is an emerging programmed cell death pathway, but its clinical relevance in HCC remains undefined. Therefore, this study aimed to identify TRPM4-associated core genes, develop and validate a prognostic signature, and investigate its relationship with the tumor immune microenvironment, tumor mutational burden, and single-cell expression patterns in HCC. METHODS: We integrated transcriptomic, clinical, and mutational datasets from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) (n=421) and Gene Expression Omnibus (GEO) cohorts (n=115) to identify genes co-expressed with TRPM4-a key NECSO mediator-and those differentially expressed in HCC. A prognostic signature was developed using least absolute shrinkage and selection operator (LASSO)-Cox regression and validated through survival analysis, time-dependent receiver operating characteristic (ROC) curves, and multivariate Cox regression analysis. The immune landscape was characterized using CIBERSORT, somatic mutation data were used to calculate tumor mutational burden (TMB) and assess its correlation with the risk score, and single-cell RNA sequencing (scRNA-seq) resolved cell-type-specific expression patterns. RESULTS: From 294 TRPM4-associated core genes, we identified an 11-gene signature (BRSK1, MMP1, GRIN2D, GP6, MYOM2, N4BP3, CCDC112, TSEN54, MAP3K9, SPP1, B3GNT4) that independently predicted overall survival (OS) (hazard ratio =5.419, P<0.001) with areas under the curve (AUCs) of 0.779, 0.693, and 0.701 at 1, 3, and 5 years. These values were superior or comparable to conventional clinicopathologic variables after direct comparison. High-risk patients exhibited an immunosuppressive microenvironment, characterized by enrichment of M0 macrophage, a higher M2/M1 ratio (P<0.001) and distinct immune checkpoint profiles. When integrated with TMB, the prognostic stratification was further refined: high-TMB/high-risk patients had poorest outcomes (median OS, 15.3 months), while low-TMB/low-risk patients had the most favorable survival (median OS, 68.7 months). Single-cell analysis revealed that MMP1 was induced in cancer-associated fibroblasts (CAFs) and SPP1 was downregulated in macrophages, single-cell risk scores confirmed TAFs and macrophages as the main contributors to the prognostic model. CONCLUSIONS: The TRPM4-centered 11-gene signature provides robust and independent prognostic stratification in HCC by integrating immune, mutational, and single-cell features. This signature serves as a potential tool for prognostic evaluation and may help inform immunotherapeutic strategies for HCC.

Hepatocellular carcinoma (HCC)

Single-nucleus profiling of postmortem diffuse midline gliomas identifies mitochondrial biogenesis as a resistance mechanism to imipridone therapy.

BACKGROUND: Imipridone ONC201 is the first FDA-approved therapy for H3K27-altered diffuse midline glioma; however, clinical responses remain limited. Defining tumor-intrinsic determinants and microenvironmental, extrinsic factors that shape sensitivity or resistance to imipridones will identify actionable therapeutic opportunities and inform improved clinical strategies. METHODS: To identify mechanisms of imipridone resistance, we obtained postmortem brain tissue from DMG patients who had received imipridones and/or standard care. Single-nucleus RNA and open-chromatin sequencing were performed on N&#x2009;=&#x2009;22 cases. Immunofluorescence-based myeloid phenotyping was performed on N&#x2009;=&#x2009;46 cases. Mitochondrial copy-number analysis was performed on N&#x2009;=&#x2009;19 cases. Validation of imipridone sensitivity, its effect on mitochondrial density, and its synergy with inhibition of mitochondrial biogenesis were assessed in DMG primary cells. RESULTS: We established a single-cell RNA/open-chromatin atlas from postmortem DMG cases and found imipridone treatment resulting in regressed mesenchymal transition, reduced myeloid-derived suppressive cells, and reversed aberrant H3K27-altered enhancer activity. Resistant tumors showed increased mitochondrial density, turnover, and membrane potential. Mitochondrial biogenesis and PPARGC1A emerged as resistance biomarkers and actionable targets. CONCLUSIONS: These studies implicate mitochondrial biogenesis as a biomarker of imipridone resistance and a focus for the development of combinatorial strategies to provide effective therapeutic options for a challenging pediatric brain tumor.

Humans