Nasopharyngeal carcinoma（NPC） is a distinct type of head and neck cancer closely associated with Epstein-Barr virus（EBV） infection and exhibits significant geographic variations in its incidence. Despite recent advancements in radiotherapy techniques and precision medicine for NPC, the overall survival rate remains unsatisfactory due to tumor metastasis, recurrence, and drug resistance. Single-cell RNA sequencing（scRNA-seq） is an emerging technology that allows for the analysis of gene expression at single-cell resolution, providing a clearer understanding of tumor cell subpopulations, the evolutionary trajectory of tumor cells, and the functional roles and interactions of cells within the tumor microenvironment. This provides new ideas for the development of precision medicine in NPC. Here, we review the applications of scRNA-seq in exploring the mechanisms of NPC pathogenesis, tumor heterogeneity, the tumor microenvironment, drug resistance, and therapeutic response.
Humans ; Nasopharyngeal Neoplasms/genetics* ; Tumor Microenvironment ; Nasopharyngeal Carcinoma ; Single-Cell Analysis ; Sequence Analysis, RNA ; Precision Medicine ; Drug Resistance, Neoplasm ; Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; Single-Cell Gene Expression Analysis

The oxytocin receptor (OXTR) has garnered increasing attention for its role in regulating both mature behaviors and brain development. It has been established that OXTR mediates a range of effects that are region-specific or period-specific. However, the current studies of OXTR expression patterns in mice only provide limited help due to limitations in resolution. Therefore, our objective was to generate a comprehensive, high-resolution spatiotemporal expression map of Oxtr mRNA across the entire developing mouse brain. We applied RNAscope in situ hybridization to investigate the spatiotemporal expression pattern of Oxtr in the brains of male mice at six distinct postnatal developmental stages (P7, P14, P21, P28, P42, P56). We provide detailed descriptions of Oxtr expression patterns in key brain regions, including the cortex, basal forebrain, hippocampus, and amygdaloid complex, with a focus on the precise localization of Oxtr⁺ cells and the variance of expression between different neurons. Furthermore, we identified some neuronal populations with high Oxtr expression levels that have been little studied, including glutamatergic neurons in the ventral dentate gyrus, Vgat⁺Oxtr⁺ cells in the basal forebrain, and GABAergic neurons in layers 4/5 of the cortex. Our study provides a novel perspective for understanding the distribution of Oxtr and encourages further investigations into its functions.
Animals ; Receptors, Oxytocin/metabolism* ; Male ; Brain/growth & development* ; Mice ; Mice, Inbred C57BL ; Neurons/metabolism* ; Single-Cell Analysis ; Gene Expression Regulation, Developmental ; RNA, Messenger/metabolism* ; Animals, Newborn

The repair of the periodontal membrane is essential for the successful management of periodontal disease and dental trauma. Emdogain^® (EMD) is widely used in periodontal therapy due to its ability to promote repair. Despite substantial research, the cellular and molecular mechanisms underlying EMD's effects, particularly at the single-cell resolution, remain incompletely understood. This study established a delayed tooth replantation model in rats to investigate these aspects. Tooth loss rate and degree of loosening were evaluated at 4 and 8 weeks. Micro-CT, HE staining, TRAP staining, and immunofluorescence staining were evaluated to assess EMD's efficacy. Single-cell sequencing analyses generated single-cell maps that explored enrichment pathways, cell communication, and potential repair mechanisms. Findings indicated that EMD could reduce the rate of tooth loss, promote periodontal membrane repair, and reduce root and bone resorption. Single-cell analysis revealed that EMD promotes the importance of Vtn+ fibroblasts, enhancing matrix and tissue regeneration functions. Additionally, EMD stimulated osteogenic pathways, reduced osteoclastic activity, and promoted angiogenesis-related pathways, particularly bone-related H-type vessel expression in endothelial cells. Gene modules associated with angiogenesis, osteogenesis, and odontoblast differentiation were identified, suggesting EMD might facilitate osteogenesis and odontoblast differentiation by upregulating endothelium-related genes. Immune cell analysis indicated that EMD did not elicit a significant immune response. Cell communication analysis suggested that EMD fostered pro-regenerative networks driven by interactions between mesenchymal stem cells, fibroblasts, and endothelial cells. In conclusion, EMD proves to be an effective root surface therapy agent that supports the restoration of delayed replantation teeth.
Animals ; Tooth Replantation/methods* ; Rats ; Dental Enamel Proteins/pharmacology* ; Single-Cell Analysis ; Rats, Sprague-Dawley ; X-Ray Microtomography ; Osteogenesis/drug effects* ; Male ; Periodontal Ligament/drug effects*

Microwave thermochemotherapy (MTC) has been applied to treat lip squamous cell carcinoma (LSCC), but a deeper understanding of its therapeutic mechanisms and molecular biology is needed. To address this, we used single-cell transcriptomics (scRNA-seq) and spatial transcriptomics (ST) to highlight the pivotal role of tumor-associated neutrophils (TANs) among tumor-infiltrating immune cells and their therapeutic response to MTC. MNDA⁺ TANs with anti-tumor activity (N1-phenotype) are found to be abundantly infiltrated by MTC with benefit of increased blood perfusion, and these TANs are characterized by enhanced cytotoxicity, ameliorated hypoxia, and upregulated IL1B, activating T&NK cells and fibroblasts via IL1B-IL1R. In this highly anti-tumor immunogenic and hypoxia-reversed microenvironment under MTC, fibroblasts accumulated in the tumor front (TF) can recruit N1-TANs via CXCL2-CXCR2 and clear N2-TANs (pro-tumor phenotype) via CXCL12-CXCR4, which results in the aggregation of N1-TANs and extracellular matrix (ECM) deposition. In addition, we construct an N1-TANs marker, MX2, which positively correlates with better prognosis in LSCC patients, and employ deep learning techniques to predict expression of MX2 from hematoxylin-eosin (H&E)-stained images so as to conveniently guide decision making in clinical practice. Collectively, our findings demonstrate that the N1-TANs/fibroblasts defense wall formed in response to MTC effectively combat LSCC.
Humans ; Neutrophils/metabolism* ; Single-Cell Analysis ; Lip Neoplasms/genetics* ; Hyperthermia, Induced/methods* ; Microwaves/therapeutic use* ; Transcriptome ; Carcinoma, Squamous Cell/immunology* ; Tumor Microenvironment

Periodontal bone defects, primarily caused by periodontitis, are highly prevalent in clinical settings and manifest as bone fenestration, dehiscence, or attachment loss, presenting a significant challenge to oral health. In regenerative medicine, harnessing developmental principles for tissue repair offers promising therapeutic potential. Of particular interest is the condensation of progenitor cells, an essential event in organogenesis that has inspired clinically effective cell aggregation approaches in dental regeneration. However, the precise cellular coordination mechanisms during condensation and regeneration remain elusive. Here, taking the tooth as a model organ, we employed single-cell RNA sequencing to dissect the cellular composition and heterogeneity of human dental follicle and dental papilla, revealing a distinct Platelet-derived growth factor receptor alpha (PDGFRA) mesenchymal stem/stromal cell (MSC) population with remarkable odontogenic potential. Interestingly, a reciprocal paracrine interaction between PDGFRA⁺ dental follicle stem cells (DFSCs) and CD31⁺ Endomucin⁺ endothelial cells (ECs) was mediated by Vascular endothelial growth factor A (VEGFA) and Platelet-derived growth factor subunit BB (PDGFBB). This crosstalk not only maintains the functionality of PDGFRA⁺ DFSCs but also drives specialized angiogenesis. In vivo periodontal bone regeneration experiments further reveal that communication between PDGFRA⁺ DFSC aggregates and recipient ECs is essential for effective angiogenic-osteogenic coupling and rapid tissue repair. Collectively, our results unravel the importance of MSC-EC crosstalk mediated by the VEGFA and PDGFBB-PDGFRA reciprocal signaling in orchestrating angiogenesis and osteogenesis. These findings not only establish a framework for deciphering and promoting periodontal bone regeneration in potential clinical applications but also offer insights for future therapeutic strategies in dental or broader regenerative medicine.
Receptor, Platelet-Derived Growth Factor alpha/metabolism* ; Humans ; Neovascularization, Physiologic/physiology* ; Dental Sac/cytology* ; Single-Cell Analysis ; Transcriptome ; Mesenchymal Stem Cells/metabolism* ; Bone Regeneration ; Animals ; Dental Papilla/cytology* ; Periodontium/physiology* ; Stem Cells/metabolism* ; Regeneration ; Angiogenesis

Smoking is a well-established risk factor for periodontitis, yet the precise mechanisms by which smoking contributes to periodontal disease remain poorly understood. Recent advances in spatial transcriptomics have enabled a deeper exploration of the periodontal tissue microenvironment at single-cell resolution, offering new opportunities to investigate these mechanisms. In this study, we utilized Visium HD single-cell spatial transcriptomics to profile gingival tissues from 12 individuals, including those with periodontitis, those with smoking-associated periodontitis, and healthy controls. Our analysis revealed that smoking disrupts the epithelial barrier integrity, induces fibroblast alterations, and dysregulates fibroblast-epithelial cell communication, thereby exacerbating periodontitis. The spatial analysis showed that endothelial cells and macrophages are in close proximity and interact, which further promotes the progression of smoking-induced periodontal disease. Importantly, we found that targeting the endothelial CXCL12 signalling pathway in smoking-associated periodontitis reduced the proinflammatory macrophage phenotype, alleviated epithelial inflammation, and reduced alveolar bone resorption. These findings provide novel insights into the pathogenesis of smoking-associated periodontitis and highlight the potential of targeting the endothelial-macrophage interaction as a therapeutic strategy. Furthermore, this study establishes an essential information resource for investigating the effects of smoking on periodontitis, providing a foundation for future research and therapeutic development for this prevalent and debilitating disease.
Humans ; Gingiva/cytology* ; Smoking/adverse effects* ; Male ; Periodontitis/pathology* ; Single-Cell Analysis ; Female ; Adult ; Middle Aged ; Macrophages ; Fibroblasts ; Endothelial Cells ; Case-Control Studies ; Chemokine CXCL12/metabolism*

Identification of disease-specific cell subtypes (DSCSs) has profound implications for understanding disease mechanisms, preoperative diagnosis, and precision therapy. However, achieving unified annotation of DSCSs in heterogeneous single-cell datasets remains a challenge. In this study, we developed the gPRINT algorithm (generalized approach for cell subtype identification with single cell's voicePRINT). Inspired by the principles of speech recognition in noisy environments, gPRINT transforms gene position and gene expression information into voiceprints based on ordered and clustered gene expression phenomena, obtaining unique "gene print" patterns for each cell. Then, we integrated neural networks to mitigate the impact of background noise on cell identity label mapping. We demonstrated the reproducibility of gPRINT across different donors, single-cell sequencing platforms, and disease subtypes, and its utility for automatic cell subtype annotation across datasets. Moreover, gPRINT achieved higher annotation accuracy of 98.37% when externally validated based on the same tissue, surpassing other algorithms. Furthermore, this approach has been applied to fibrosis-associated diseases in multiple tissues throughout the body, as well as to the annotation of fibroblast subtypes in a single tissue, tendon, where fibrosis is prevalent. We successfully achieved automatic prediction of tendinopathy-specific cell subtypes, key targets, and related drugs. In summary, gPRINT provides an automated and unified approach for identifying DSCSs across datasets, facilitating the elucidation of specific cell subtypes under different disease states and providing a powerful tool for exploring therapeutic targets in diseases.
Humans ; Algorithms ; Single-Cell Analysis ; Databases, Genetic ; Molecular Sequence Annotation

Dysregulated RNA splicing is a well-recognized characteristic of colorectal cancer (CRC); however, its intricacies remain obscure, partly due to challenges in profiling full-length transcript variants at the single-cell level. Here, we employ high-depth long-read scRNA-seq to define the full-length transcriptome of colorectal epithelial cells in 12 CRC patients, revealing extensive isoform diversities and splicing alterations. Cancer cells exhibited increased transcript complexity, with widespread 3'-UTR shortening and reduced intron retention. Distinct splicing regulation patterns were observed between intrinsic-consensus molecular subtypes (iCMS), with iCMS3 displaying even higher splicing factor activities and more pronounced 3'-UTR shortening. Furthermore, we revealed substantial shifts in isoform usage that result in alterations of protein sequences from the same gene with distinct carcinogenic effects during tumorigenesis of CRC. Allele-specific expression analysis revealed dominant mutant allele expression in key oncogenes and tumor suppressors. Moreover, mutated PPIG was linked to widespread splicing dysregulation, and functional validation experiments confirmed its critical role in modulating RNA splicing and tumor-associated processes. Our findings highlight the transcriptomic plasticity in CRC and suggest novel candidate targets for splicing-based therapeutic strategies.
Humans ; Colorectal Neoplasms/metabolism* ; RNA Isoforms/metabolism* ; Single-Cell Analysis ; RNA Splicing ; Gene Expression Regulation, Neoplastic ; RNA, Neoplasm/metabolism* ; Transcriptome

Ancient traditional Chinese medicine (TCM) doctrine says "The superior doctor prevents illnesses," pointing out preventative medicine as the ultimate goal for medical care. TCM recognizes that genetic predisposition and environmental and lifestyle influences contribute to diseases. It divides people into eight constitutions in addition to one normal/healthy kind. People with one of the eight subhealth constitutions are prone to develop different kinds of corresponding illnesses. The goal for this type of categorization is to help people take preemptive measures to prevent or delay disease onset. As the peripheral immune system through surveying the body, it can capture information from essentially all organs and reflect anomalies occurring in each organ. Thus, the detailed profiling of the peripheral immune-system function can generally reflect a person's overall heath state. In this study, we performed the single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from individuals with Tanshi (phlegm dampness) constitution. They were prone to develop metabolic disorders including diabetes. scRNA-seq revealed greatly reduced mucosal-associated invariable T cell content and heightened TNFα-NFκB, JAK-STAT, and interferon signaling. These findings indicated heightened chronic inflammation, as well as increased hypoxia/apoptosis responses, likely resulting from frequent sleep apnea that Tanshi individuals experienced. Altogether, this pilot study demonstrated effectiveness in using scRNA-seq to reveal molecular-immunological bases for constitution categorization, thereby substantiating that preventative medicine originated from TCM.
Humans ; Leukocytes, Mononuclear/metabolism* ; Male ; Female ; Gene Expression Profiling ; Single-Cell Analysis ; Middle Aged ; Adult ; Medicine, Chinese Traditional ; Transcriptome ; Single-Cell Gene Expression Analysis

OBJECTIVE: To identify prognostic genes associated with lysosome-dependent cell death (LDCD) in patients with gastric cancer (GC). METHODS: Differentially expressed genes (DEGs) were identified using The Cancer Genome Atlas - Stomach Adenocarcinoma. Weighted gene co-expression network analysis was performed to identify the key module genes associated with LDCD score. Candidate genes were identified by DEGs and key module genes. Univariate Cox regression analysis, and least absolute shrinkage and selection operator regression and multivariate Cox regression analyses were performed for the selection of prognostic genes, and risk module was established. Subsequently, key cells were identified in the single-cell dataset (GSE183904), and prognostic gene expression was analyzed. Cell proliferation and migration were assessed using the Cell Counting Kit-8 assay and the wound healing assay. RESULTS: A total of 4,465 DEGs, 95 candidate genes, and 4 prognostic genes, including C19orf59, BATF2, TNFAIP2, and TNFSF18, were identified in the analysis. Receiver operating characteristic curves indicated the excellent predictive power of the risk model. Three key cell types (B cells, chief cells, and endothelial/pericyte cells) were identified in the GSE183904 dataset. C19orf59 and TNFAIP2 exhibited predominant expression in macrophage species, whereas TNFAIP2 evolved over time in endothelial/pericyte cells and chief cells. Functional experiments confirmed that interfering with C19orf59 inhibited proliferation and migration in GC cells. CONCLUSION C19orf59, BATF2, TNFAIP2, and TNFSF18 are prognostic genes associated with LDCD in GC. Furthermore, the risk model established in this study showed robust predictive power.
Stomach Neoplasms/pathology* ; Humans ; Prognosis ; Lysosomes/physiology* ; RNA-Seq ; Cell Death ; Single-Cell Analysis ; Gene Expression Regulation, Neoplastic ; Cell Proliferation ; Single-Cell Gene Expression Analysis