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Metabolic reprogramming and M2 macrophage depletion define the microenvironment of adenomyosis.
BACKGROUND: Adenomyosis is a chronic gynecological disorder characterized by the invasion of endometrial tissues into the myometrium of the uterus, with pathophysiology linked to chronic inflammation and metabolic dysregulation. However, the understanding of the molecular mechanisms and underlying pathologies remains limited. This study aims to elucidate the metabolic reprogramming and immune dysregulation within the eutopic endometrium from patients diagnosed with adenomyosis, and identify potential therapeutic targets or diagnostic indicators. METHODS: We analyzed publicly available microarray (GSE78851), bulk RNA-seq (GSE193928) and single-cell RNA-seq (Human Endometrial Cell Atlas) datasets to explore the microenvironment of eutopic endometrium from adenomyosis patients. Transcriptomic differences were assessed in GSE78851 (3 adenomyosis versus 5 controls), followed by immune composition and metabolic flux analysis. Findings on immune infiltration and metabolic changes were further validated in GSE193928 (6 adenomyosis versus 15 controls after quality control). Metabolic flux analysis was further extended to single-cell RNA sequencing data derived from endometrial samples of 63 donors. Key genes involved in keratan sulfate biosynthesis were further validated by RT-qPCR and immunofluorescence staining on endometrial biopsy samples. RESULTS: Our study revealed widespread reprogramming in eutopic adenomyosis endometrium, characterized by enhanced immune-related pathways, reduction of M2 macrophage abundance, and disrupted metabolic processes. Further investigation of scRNA-seq data highlighted the cell type-specific metabolic profiles and immune-metabolic interplay within the endometrial microenvironment. Notably, the dysfunction of keratan sulfate biosynthesis, coupled with reduced M2 macrophage level, emerged as a consistent feature. Importantly, four key genes involved in keratan sulfate biosynthesis, including CHST1, CHST6, B4GALT1, and B3GNT2, were upregulated in eutopic endometrium compared to controls, suggesting their potential role in the pathophysiology of the disease. CONCLUSIONS: This study identifies dysregulation of keratan sulfate biosynthesis as a central feature of adenomyosis and links it to reduced M2 macrophage abundance and immune-metabolic imbalance. Validation of four keratan sulfate related genes strengthens their potential as biomarkers or therapeutic targets, providing novel mechanistic insight into the pathogenesis of adenomyosis.
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Oncolytic Virus Infection Modulates Lysine Acetyltransferase in Gliomas: Comprehensive Analysis and Experimental Validation of KAT8 in Glioma.
Oncolytic virotherapy, which uses engineered viruses to selectively target tumour cells, has emerged as a potential treatment in glioma. However, how oncolytic virus infection modulates lactylation enzymes in gliomas remains unclear. The RNA-seq data after oncolytic virus EV-A71 infection on glioma cells was analysed to screen and lysine acetyltransferase 8 (KAT8) was selected. We analysed KAT8 expression in glioma tissues using data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Associations between KAT8 expression and clinicopathological features were evaluated. Kaplan-Meier survival analysis and Cox regression models were used to assess the prognostic value of KAT8. Functional annotation analyses were performed to explore the KAT8-related biological processes. Single-cell RNA-sequencing data were analysed to investigate cell-type-specific KAT8 expression. In vitro experiments were conducted to validate the effects of KAT8 knockdown on apoptosis in glioma cells. KAT8 was among the top 5 down-regulated genes in glioma cells after oncolytic viruses EV-A71 infection. KAT8 was significantly overexpressed in glioma tissues compared to normal brain tissues, with higher expression in lower-grade gliomas. High KAT8 expression was associated with 1p19q co-deletion, IDH mutation, and younger patient age. Notably, high KAT8 expression correlated with better overall survival in grade IV gliomas. Functional analyses revealed KAT8's involvement in neuronal development, mRNA processing, and apoptosis regulation. Single-cell sequencing data revealed a negative correlation between KAT8 expression and apoptosis in glioblastoma cells. In vitro experiments confirmed the increased apoptosis in glioma cell lines following KAT8 knockdown. Our findings suggest that the regulatory pathway of oncolytic virus infection for lactylation enzymes may be a key pathway affecting gliomas. These findings highlight KAT8 as a potential prognostic biomarker and therapeutic target for gliomas.
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Asian diversity in human immune cells.
The relationships of human diversity with biomedical phenotypes are pervasive yet remain understudied, particularly in a single-cell genomics context. Here, we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 donors, spanning 7 population groups across 5 Asian countries, and 6 controls. Though population groups are frequently compared at the continental level, we found that sub-continental diversity, age, and sex pervasively impacted cellular and molecular properties of immune cells. These included differential abundance of cell neighborhoods as well as cell populations and genes relevant to disease risk, pathogenesis, and diagnostics. We discovered functional genetic variants influencing cell-type-specific gene expression, which were under-represented in non-Asian populations, and helped contextualize disease-associated variants. AIDA enables analyses of multi-ancestry disease datasets and facilitates the development of precision medicine efforts in Asia and beyond.
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Epigenetic inactivation of COL4A4 orchestrates JAK2/STAT3-driven progression and microenvironment reprogramming in renal cell carcinoma.
BACKGROUND: Renal cell carcinoma (RCC) is a common urinary malignancy that lacks reliable early biomarkers. Although epigenetic silencing of tumor suppressor genes mediated by DNA methylation is common in RCC, the specific role and regulation of the basement membrane component COL4A4 remain unclear. METHODS: Bulk and single-cell RNA sequencing (scRNA-seq), DNA methylation analysis, The Cancer Genome Atlas data and in-house clinical RCC cohorts were integrated to characterize COL4A4 expression, epigenetic regulation, clinical significance, and associations with tumor microenvironment (TME) features. The biological functions of COL4A4 were evaluated using in vitro functional assays and in vivo xenograft models. Mechanistic studies included 5-Aza demethylation treatment and JAK2/STAT3 pathway inhibition. RESULTS: COL4A4 was downregulated in RCC, and its downregulation is associated with promoter hypermethylation, serving as an independent prognostic factor. Functionally, COL4A4 inhibited RCC cell proliferation, migration, and invasion in vitro, and suppressed tumor growth in vivo. Mechanistically, COL4A4 negatively modulated the JAK2/STAT3 signaling pathway. scRNA-seq revealed that COL4A4 was predominantly enriched in epithelial cells. Low COL4A4 expression is associated with altered extracellular matrix-receptor interactions and an immunosuppressive TME. CONCLUSIONS: COL4A4 acts as a methylation-silenced RCC tumor suppressor that negatively modulates JAK2/STAT3 signaling and correlates with TME features, representing a potential epigenetic therapeutic target.
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A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.
Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.
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A novel in vitro model of primary human pediatric lung epithelial cells.
BACKGROUND: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS: PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.
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The prognostic significance of KLRB1 and its further association with immune cells in breast cancer.
BACKGROUND: Killer cell lectin-like receptor B1 (KLRB1) is an important member of the natural killer cell gene family. This study explored the potential value of KLRB1 as a breast cancer (BC) biomarker and its close association with the tumor immune microenvironment during the development of BC. METHODS: We examined the differential expression of KLRB1 in pan-cancer. Clinical and RNA-Seq data from BC samples were evaluated in The Cancer Genome Atlas (TCGA) and validated in Gene Expression Omnibus (GEO) datasets and by immunohistochemistry (IHC) staining. The relationship between KLRB1 and clinical parameters was explored through Chi-square tests. The diagnostic value of KLRB1 was evaluated using a receiver operating characteristic (ROC) curve. Survival analysis was tested by Kaplan-Meier curves to demonstrate the relationship between KLRB1 and survival. Univariable and multivariate cox regression analyses were carried out as well. The analysis of immune infiltration level and gene set enrichment analysis (GSEA) were conducted to examine KLRB1's mechanism during the progression of BC. We used the Tumor Immune Estimation Resource (TIMER), the Cancer Single-cell Expression Map (CancerSCEM) database, the Tumor Immune Single-cell Hub (TISCH) database, and the Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) method to explore KLRB1's association with immune infiltration level and different quantitative distribution of immune cells. The relevant signaling pathways in BC associated with KLRB1 were identified using GSEA. RESULTS: The expression of KLRB1 was downregulated across the majority of cancers including BC. The lower KLRB1 expression group exhibited shorter relapse free survival (RFS) and overall survival (OS). IHC staining showed that KLRB1 staining was weaker in breast tumor tissues than in paratumors. Additionally, GSEA identified several pathway items distinctly enriched in BC. KLRB1 expression level was also positively related to the infiltrating number of immune cells in BC. Moreover, the CancerSCEM and TISCH databases as well as the CIBERSORT method demonstrated the close relationship between KLRB1 and immune cells, particularly macrophages. CONCLUSION: Low KLRB1 expression was considered an independent prognostic biomarker and played an important role in the tumor immune microenvironment of BC patients.
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Integrative Multi-Omics and Machine Learning Reveal Senescent Fibroblast-Associated ADAMTS2 as a Critical Indicator of Intestinal Inflammation in IBD.
Although cellular senescence contributes to tissue dysfunction in inflammatory bowel disease (IBD), the heterogeneity of senescence at the single-cell resolution remains poorly defined. In this study, we systematically characterized the senescence landscape within the IBD microenvironment, with particular emphasis on fibroblast subpopulations and their systemic consequences. Single-cell RNA sequencing (scRNA-seq) dataset was applied to profile senescence-associated feature heterogeneity. Fibroblasts were classified into high-senescence (HS-Fibro) and low-senescence (LS-Fibro) groups according to senescence scores. HS-Fibro-specific gene signatures were identified and functionally annotated. Intercellular communication networks were constructed using CellChat to delineate the HS-Fibro interactome atlas, with emphasis on identifying cellular targets responsive to senescence-associated secretory phenotype (SASP) signals. Integrated microarray datasets were employed to elucidate the systemic impact of HS-Fibro in IBD. Through the integration of differential expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), and an ensemble of 113 machine learning algorithms, seven hub genes implicated in IBD progression were systematically identified and prioritized. In the IBD microenvironment, fibroblasts exhibited enhanced senescence activity. HS-Fibro displayed potent pro-inflammatory properties and enhanced communicative capacity. IBD patients with high HS-Fibro infiltration showed sustained immune-inflammatory activity. Systematic bioinformatic analysis and immunohistochemistry validation identified ADAMTS2 as a robust diagnostic indicator for IBD, with substantially elevated expression in patients relative to controls. The human colon fibroblast cell line CCD-18Co was treated with H2O2 to establish a cellular senescence model, demonstrating marked upregulation of ADAMTS2, SASP markers, and fibrosis-related genes. Moreover, siRNA-mediated knockdown of ADAMTS2 resulted in obvious attenuation of these SASP and fibrosis-associated genes.
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Etiology-based Molecular Characterization of Hepatocellular Carcinoma Reveals SQLE's Contribution to Immunosuppressive Microenvironment.
BACKGROUND: Hepatocellular carcinoma (HCC) is a kind of fatal cancer with a variety of risk factors. However, a pan-etiology molecular characterization of HCC has not been explored. OBJECTIVE: The objective of this study is to explore etiology-specific features of HCC and improve our understanding of tumorigenesis, thereby revealing potential therapeutic targets. METHODS: RNA-seq, genetic alteration, copy number alteration, and clinicopathological data of The Cancer Genome Atlas -Liver Hepatocellular Carcinoma cohort were downloaded and explored. Immune-related features and single-cell and spatial transcriptomic data were adopted and analyzed. RESULTS: Etiology-based analyses revealed that HCC with different etiologies showed different clinical features, including gender composition, ethnic composition, clinical stage distribution, and survival. In addition, distinct genetic alterations, copy number alterations, and tumor microenvironment were observed in HCC patients with different etiologies. Significantly enhanced expression of squalene epoxidase (SQLE) was observed in viral hepatitis-related HCC and was associated with poor tumor grade and overall survival. Correlation analysis revealed a negative relationship between SQLE expression and anti-tumor immunity. Single-cell and spatial transcriptomics demonstrated that SQLE contributed to reduced T cell and NK cell infiltration while increasing macrophage and monocyte infiltration. CONCLUSION: The current study demonstrated that HCC has etiology-specific clinical features, genetic alteration, copy number alteration, and tumor microenvironment. Overexpression of SQLE in viral hepatitis-related HCC correlate with poor clinical outcome and may contribute to reduce T cell and NK cell infiltration while increased macrophage and monocyte infiltration, which lead to immunosuppressive microenvironment and can be an actionable target.
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A Single-Cell Transcriptome Profiling of Triptolide-Induced Nephrotoxicity in Mice.
Triptolide (TP), an active component isolated from the traditional Chinese herb Tripterygium wilfordii Hook F (TWHF), shows great promise for treating inflammation-related diseases. However, its potential nephrotoxic effects remain concerning. The mechanism underlying TP-induced nephrotoxicity is inadequately elucidated, particularly at single-cell resolution. Hence, single-cell RNA sequencing (scRNA-seq) of kidney tissues from control and TP-treated mice is performed to generate a thorough description of the renal cell atlas upon TP treatment. Heterogeneous responses of nephron epithelial cells are observed after TP exposure, attributing differential susceptibility of cell subtypes to excessive reactive oxygen species and increased inflammatory responses. Moreover, TP disrupts vascular function by activating endothelial cell immunity and damaging fibroblasts. Severe immune cell damage and the activation of pro-inflammatory Macro_C1 cells are also observed with TP treatment. Additionally, ligand-receptor crosstalk analysis reveals that the SPP1 (osteopontin) signaling pathway targeting Macro_C1 cells is triggered by TP treatment, which may promote the infiltration of Macro_C1 cells to exacerbate renal toxicity. Overall, this study provides comprehensive information on the transcriptomic profiles and cellular composition of TP-associated nephrotoxicity at single-cell resolution, which can strengthen the understanding of the pathogenesis of TP-induced nephrotoxicity and provide valuable clues for the discovery of new therapeutic targets to ameliorate TP-associated nephrotoxicity.