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Single-cell RNA sequencing of a poorly metastatic melanoma cell line and its subclones with high lung and brain metastasis potential reveals gene expression signature of metastasis with prognostic implication.
The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.
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FGMD: A novel approach for functional gene module detection in cancer.
With the increasing availability of multi-dimensional biological datasets for the same samples (i.e., gene expression, microRNAs, copy numbers, mutations, methylations), it has now become possible to systematically understand the regulatory mechanisms operating in a cancer cell. For this task, it is important to discover a set of co-expressed genes with functions, representing a so-called functional gene module, because co-expressed genes tend to be co-regulated by the same regulators, including transcription factors, microRNAs, and copy number aberrations. Several algorithms have been used to identify such gene modules, including hierarchical clustering and non-negative matrix factorization. Although these algorithms have been applied to many microarray datasets, only a few systematic analyses of these algorithms have been performed for RNA-sequencing (RNA-Seq) data to date. Although gene expression levels determined based on microarray and RNA-Seq datasets tend to be highly correlated, the expression levels of some genes differ depending on the platforms used for analysis, which may result in the construction of different gene modules for the same samples. Here, we compare several module detection algorithms applied to both microarray and RNA-seq datasets. We further propose a new functional gene module detection algorithm (FGMD), which is based on a hierarchical clustering algorithm that was modified to reflect actual biological observations, including the fact that a single gene may be involved in multiple biological pathways. Application of existing algorithms and the new FGMD algorithm to breast cancer and ovarian cancer datasets from The Cancer Genome Atlas showed that the FGMD algorithm had the best performance for most of the functional pathway enrichment tests and in the transcription factor enrichment test. We expect that the FGMD algorithm will contribute to improving the identification of functional gene modules related to cancer.
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A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands.
Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse, we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos population express the hallmarks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2bpos ductal and myoepithelial cells.
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Metabolomic and Single-Cell Transcriptomic Analyses Shed Light on Secondary Metabolite Profiling and Potential Developmental Dynamics of Glandular Trichomes in Artemisia argyi.
Plant glandular trichomes (GTs) are characterised by their ability to synthesise and store abundant secondary metabolites of significant biological importance. The medicinal plant Artemisia argyi exhibits a dense covering of GTs, yet the precise metabolic compositions and the developmental molecular dynamics of A. argyi GTs remain insufficiently characterised. Using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), we identified 969 differentially accumulated metabolites (DAMs) between GTs and non-glandular trichomes (NGTs) of A. argyi. GTs were significantly enriched in diverse metabolites, including terpenoids, flavonoids and fatty acyls, among which sesquiterpenoids were the most abundant terpenoid subclass in DAMs. To elucidate the molecular basis of GT secondary metabolite biosynthesis and development, we generated a single-cell transcriptomic atlas of A. argyi leaves, annotating cell populations of mesophyll cells (MCs), epidermal cells (ECs), vascular cells (VCs), stomatal guard cells (GCs) and GTs. Pseudotime trajectory analysis uncovered the continuous developmental trajectory of GTs and identified several candidate transcription factors (TFs) potentially involved in GT development. Leveraging the single-cell atlas, we constructed cell-type-specific co-expression networks for sesquiterpene biosynthesis genes. Within the GT-specific expression module, we identified β-caryophyllene synthase AarTPS77, while the EC-specific AarTPS52 was found to function as β-farnesene synthase. Furthermore, we functionally characterised AarTPS95 and AarTPS96, which catalyse the formation of germacrene A and 12 additional sesquiterpenoids. These findings provide insights into the molecular basis of trichome development and secondary metabolite accumulation in A. argyi, laying the foundation for improving the quality of medicinal materials.
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Single-cell analysis of matrisome-related genes in breast invasive carcinoma: new avenues for molecular subtyping and risk estimation.
BACKGROUND: The incidence of breast cancer remains high and severely affects human health. However, given the heterogeneity of tumor cells, identifying additional characteristics of breast cancer cells is essential for accurate treatment. PURPOSE: This study aimed to analyze the relevant characteristics of matrix genes in breast cancer through the multigroup data of a breast cancer multi-database. METHODS: The related characteristics of matrix genes in breast cancer were analyzed using multigroup data from the breast cancer multi database in the Cancer Genome Atlas, and the differential genes of breast cancer matrix genes were identified using the elastic net penalty logic regression method. The risk characteristics of matrix genes in breast cancer were determined, and matrix gene expression in different breast cancer cells was evaluated using real-time fluorescent quantitative polymerase chain reaction (PCR). A consensus clustering algorithm was used to identify the biological characteristics of the population based on the matrix molecular subtypes in breast cancer, followed by gene mutation, immune correlation, pathway, and ligand-receptor analyses. RESULTS: This study reveals the genetic characteristics of cell matrix related to breast cancer. It is found that 18.1% of stromal genes are related to the prognosis of breast cancer, and these genes are mostly concentrated in the biological processes related to metabolism and cytokines in protein. Five different matrix-related molecular subtypes were identified by using the algorithm, and it was found that the five molecular subtypes were obviously different in prognosis, immune infiltration, gene mutation and drug-making gene analysis. CONCLUSIONS: This study involved analyzing the characteristics of cell-matrix genes in breast cancer, guiding the precise prevention and treatment of the disease.
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Curcumin modulates the function of SPP1+ macrophages via NF-κB signaling to alleviate endometriosis.
Endometriosis is a complex gynecological disorder with prominent cellular heterogeneity, and its key pathogenic cell subsets and core molecular mechanisms remain elusive, which restricts the development of effective therapeutic strategies. In this study, we constructed a single-cell atlas of endometriotic lesions via single-cell RNA sequencing, combined with multi-omics analysis, cell-cell communication mining, functional validation and animal experiments. We identified SPP1+ macrophages as the key pathogenic macrophage subset in endometriosis, and uncovered its core mechanism of action: SPP1+ macrophages, as the most significantly enriched core differential cell type in endometriotic lesions, underwent pro-inflammatory reprogramming by activating the NF-κB signaling pathway, thereby regulating the inflammatory microenvironment in lesion sites. Meanwhile, SPP1+ macrophages served as central hubs in the dysregulated signaling network, mediating paracrine crosstalk with stromal and epithelial cells through the SPP1 signaling pathway to drive the pathogenesis of endometriosis. Furthermore, we clarified a novel molecular mechanism underlying curcumin-mediated alleviation of endometriosis: curcumin targeted the NF-κB signaling pathway of SPP1+ macrophages, inhibited NF-κB activation via stable binding to p65, thereby downregulating NFKB1 expression and suppressing SPP1-triggered inflammatory programming, ultimately regulating the inflammatory phenotype of macrophages. Animal experiments suggested that curcumin significantly reduced the volume of ectopic endometriotic lesions and downregulated NFKB1 expression in lesion tissues. This study identified the SPP1+ macrophage/NF-κB signaling axis as a critical driver of endometriosis pathogenesis, and provided novel experimental evidence and molecular mechanistic support for curcumin in the treatment of endometriosis by targeting this axis, laying an important theoretical foundation for the development of targeted therapies for endometriosis.
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A Single-Cell Atlas of DNA Methylation in Autism Spectrum Disorder Reveals Distinct Regulatory and Aging Signatures.
UNLABELLED: Autism spectrum disorder (ASD) is a common, genetically and clinically heterogeneous neurodevelopmental condition. Despite this diversity, studies of postmortem brain tissue have revealed convergent molecular changes across the cortex, including reduced synaptic function in subsets of excitatory and inhibitory neurons and increased glial reactivity. Whether these features are reflected in cell type-specific epigenetic signatures remains unknown. Here, we present the first single-cell analysis of DNA methylation (DNAm) coupled with transcriptomics in ASD. Using snmCT- seq, we profiled DNAm and gene expression from over 60,000 nuclei across 49 donors. We identified thousands of differentially methylated regions (DMRs) in ASD, enriched in promoters and regulatory elements active during both prenatal development and in adult cortex. ASD-related methylation changes were spatially localized but uncorrelated with gene expression, and were small in magnitude compared to robust age-associated effects. Age-DMRs were concentrated in excitatory neurons, enriched in known cognitive aging pathways, and revealed distinct roles for CG and non- CG methylation in the aging brain. Finally, we explored age-by-diagnosis interactions, identifying a reduction in inhibitory neuron abundance with age in ASD relative to controls, highlighting this area as a promising direction for future research. HIGHLIGHTS: We generate a single cell multi-omic dataset, jointly profiling DNA methylation and gene expression in autistic and neurotypical donorsWe identify thousands of cell type informed differentially methylated regions (DMRs) in ASD, particularly in excitatory neurons from superficial cortical lamina and microgliaASD-DMRs are enriched in promoters and known regulatory regions, but not strongly tied to gene expressionAge effects on DNA methylation are profound, cell type specific, and concentrated in excitatory neurons.
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CD8+ T-Cell Signatures as Prognostic and Immunotherapy Response Predictors in Non-Small Cell Lung Cancer.
Non-small cell lung carcinoma (NSCLC) represents the majority of lung cancer cases, comprising approximately 85 % of the total. The five-year survival rate for NSCLC patients remains discouragingly low. Recently, immunotherapy has emerged as a promising approach. Nevertheless, only a minority of patients experience considerable benefits from these treatments. This highlights the critical need for effective biomarkers that can predict both patient prognosis and response to immunotherapy. CD8+ T cells play a crucial role in cancer immunotherapy. Their presence within tumours is generally indicative of a favourable prognosis and increased efficacy of immunotherapy. This study was undertaken to identify and authenticate a novel biomarker signature based on CD8+ T-cell marker genes, to prognosticate therapeutic responses in individuals afflicted with NSCLC. This in-depth study was based on a total of 1,200 samples, which included four NSCLC specimens analysed through single-cell RNA sequencing (scRNA-seq), 1,000 NSCLC samples obtained from The Cancer Genome Atlas (TCGA) and 196 NSCLC specimens collected from the GSE37745 cohort. In patients with NSCLC, those presenting a favourable risk profile demonstrated notable elevations in specific immune cells while concurrently exhibiting reductions in other types. CD8+ T cells, with their established role in inducing apoptosis in cancer cells, have emerged as crucial predictors and modulators of treatment strategies for NSCLC patients. The combination of single-cell and bulk RNA sequencing has produced a biomarker signature, emphasizing the CD8+ T cells' crucial role in NSCLC prognosis and treatment.
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An atlas of early human mandibular endochondral and osteogenic paracrine signaling regions of Meckel's cartilage.
The mandible, also known as the lower jaw, is the only bone in the skull that can move and is essential for speaking and chewing. Meckel's cartilage (MC) is a temporary structure that supports the formation of the mandible, but how MC is involved in the ossification of the mandible is poorly understood. Through the use of single-cell RNA sequencing and single-cell spatial transcriptomics analyses, a spatiotemporal atlas of MC in human fetuses from 7 to 15 wk postconception was established, highlighting the role of MC in the ossification of the mandible. Importantly, we revealed that two populations of MC contributed to mandibular ossification through different mechanisms. The anterior MC can differentiate into osteolineage cells, as shown in an in vivo lineage tracing mouse model. The intermediate MC facilitates intramembranous ossification through cell-cell communications, possibly through signaling ligands like BMP5, BMP7, SEMA3A, PDGFC, and FGF7. This study suggests that MC plays a crucial role in mediating mandibular ossification through distinct mechanisms, providing valuable insights for understanding oral and craniofacial diseases and disorders in the future.
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Single-nucleus mRNA-sequencing reveals dynamics of lipogenic and thermogenic adipocyte populations in murine brown adipose tissue in response to cold exposure.
OBJECTIVE AND METHODS: Brown adipose tissue (BAT) comprises a heterogeneous population of adipocytes and non-adipocyte cell types. To characterize these cellular subpopulations and their adaptation to cold, we performed single-nucleus mRNA-sequencing (snRNA-seq) on interscapular BAT from mice maintained at room temperature or exposed to acute (24h) or chronic (10 days) cold (6 °C). To investigate the role of the de novo lipogenesis (DNL)-regulating transcription factor carbohydrate response element-binding protein (ChREBP), we analyzed control and brown adipocyte-specific ChREBP knockout mice. RESULTS: We identified different cell populations, including seven brown adipocyte subtypes with distinct metabolic profiles. One of them highly expressed ChREBP and DNL enzymes. Notably, these lipogenic adipocytes were highly sensitive to acute cold exposure, showing a marked depletion in BAT of control mice that was compensated by other brown adipocyte subtypes maintaining DNL. Chronic cold exposure resulted in an expansion of basal brown adipocytes and adipocytes putatively derived from stromal and endothelial precursors. In ChREBP-deficient mice, lipogenic adipocytes were almost absent under all conditions, identifying the transcription factor as a key determinant of this adipocyte subtype. Detailed expression analyses revealed Ttc25 as a specific marker of lipogenic brown adipocytes and as a downstream target of ChREBP. Furthermore, pathway and cell-cell interaction analyses implicated a Wnt-ChREBP axis in the maintenance of lipogenic adipocytes, with Wnt ligands from stromal and muscle cells providing instructive cues. CONCLUSIONS: Our findings provide a comprehensive atlas of BAT cellular heterogeneity and reveal a critical role for ChREBP in lipogenic adipocyte identity, with implications for BAT plasticity and metabolic function.