Single-cell Sequencing Research Articles

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies.

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

A novel protein encoded by porcine circANKRD17 activates the PPAR pathway to regulate intramuscular fat metabolism.

Intramuscular fat is an important factor in evaluating pork quality and varies widely among different pig breeds. However, the regulatory mechanism of circular RNAs (circRNAs) in lipid metabolism remains largely unexplored. We combined circRNA-seq and Ribo-seq data to screen a total of 18 circRNA candidates with coding potential, and circANKRD17 was found to be significantly elevated in the longissimus dorsi muscle of Lantang piglets, with a length of 1,844 nucleotides. Using single-cell sequencing, we identified 477 differentially expressed genes in IMF cells between Lantang and Landrace piglets, with enrichment in the PPAR signaling pathway. These genes included FABP4, FABP5, CPT1A, and UBC, consistent with the high levels of acylcarnitines observed in the longissimus dorsi muscles of the Lantang breed, as determined by lipidomic analysis. Further in vitro and in vivo experiments indicated that circANKRD17 can regulate lipid metabolism through various mechanisms involving the PPAR pathway, including promoting adipocyte differentiation, fatty acid transport and metabolism, triglyceride synthesis, and lipid droplet formation and maturation. In addition, we discovered that circANKRD17 has an open reading frame and can be translated into a novel 571-amino-acid protein that promotes lipid metabolism. Our research provides new insights into the role of protein-coding circANKRD17, especially concerning the metabolic characteristics of pig breeds with higher intramuscular fat content.

Identification of mitochondrial carrier homolog 2 as an important therapeutic target of castration-resistant prostate cancer

Abstract We here investigate the expression of the mitochondrial carrier homolog 2 (MTCH2) and its potential function in castration-resistant prostate cancer (CRPC). Bioinformatic analyses reveal that MTCH2 overexpression is associated with critical clinical parameters of prostate cancer. Single-cell sequencing data indicate elevated MTCH2 expression in the prostate cancer epithelium. MTCH2 is also upregulated in locally treated CRPC tissue and various primary human CRPC cells. Using genetic silencing via shRNA and knockout (KO) through the CRISPR-sgRNA approach, we showed that the depletion of MTCH2 impaired mitochondrial function, resulting in a reduced oxygen consumption rate, diminished complex I activity, and decreased ATP levels, mitochondrial depolarization, and increased reactive oxygen species production in primary CRPC cells. The silencing or KO of MTCH2 significantly inhibited cell viability, proliferation, and migration, together with a marked increase in apoptosis in the primary CRPC cells. In contrast, ectopic expression of MTCH2 provided CRPC cells with pro-tumorigenic properties, enhancing ATP production and promoting cell proliferation and migration. MTCH2 silencing also markedly inhibited the growth of subcutaneous xenografts of the primary CRPC cells in nude mice. The MTCH2-silenced xenografts exhibited increased apoptosis, elevated lipid peroxidation, and decreased ATP levels. These results provide new insights into the role of MTCH2 in supporting mitochondrial function and CRPC progression.

Usp11 maintained the survival of marginal zone B cells under ionizing radiation by deubiquitinating DLL1 and JAG2.

Efficacy of radiation therapy is compromised by hematopoietic and immune impairments, with elusive underlying causes. This study aimed to elucidate Usp11's role in radiation-induced injuries and uncover related mechanisms. Utilized ARS mouse model to observe survival rates of Usp11-/- (KO) mice post-TBI (Total Body Irradiation). Assessed lymphocyte and MZ B (Marginal Zone B) cell rates using histological analysis, single-cell sequencing, immunofluorescence (IF), immunohistochemistry (IHC), and flow cytometry (FCM). Conducted Co-IP and ubiquitination experiments for mechanism elucidation. Quantified IgM and IgG using ELISA and FC. Explored public databases for potential correlation molecules. Our findings indicated that Usp11-/- mice exhibited improved survival rates following TBI, with the spleen playing a pivotal role. HE staining revealed a wider marginal zone in the spleen of Usp11+/+ mice post-irradiation. Single-cell sequencing, IF, IHC, and FCM analyses revealed a higher survival rate of MZ B cells in Usp11-/- mice after irradiation. Furthermore, treatment with the Usp11 inhibitor, mitoxantrone, successfully targeted and inhibited Usp11, thereby alleviating the reduction in MZ B cells in the spleen following total body irradiation. Mechanistically, Usp11 sustained the survival of MZ B cells by regulating the ubiquitination of Notch's ligands, DLL1 and JAG2, thereby promoting immune cell remodeling in the spleen. In conclusion, Usp11 played a crucial role in modulating immune system damage induced by ionizing radiation, primarily through ubiquitination of Notch ligands. This study provides insights into radiation-induced immune injuries and suggests Usp11 as a potential therapeutic target.

O-GlcNAcylation attenuates ischemia-reperfusion-induced pulmonary epithelial cell ferroptosis via the Nrf2/G6PDH pathway

BackgroundLung ischemia–reperfusion (I/R) injury is a common clinical pathology associated with high mortality. The pathophysiology of lung I/R injury involves ferroptosis and elevated protein O-GlcNAcylation levels, while the effect of O-GlcNAcylation on lung I/R injury remains unclear. This research aimed to explore the effect of O-GlcNAcylation on reducing ferroptosis in pulmonary epithelial cells caused by I/R.ResultsFirst, we identified O-GlcNAc transferase 1 (Ogt1) as a differentially expressed gene in lung epithelial cells of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients, using single-cell sequencing, and Gene Ontology analysis (GO analysis) revealed the enrichment of the ferroptosis process. We found a time-dependent dynamic alteration in lung O-GlcNAcylation during I/R injury. Proteomics analysis identified the differentially expressed proteins enriched in ferroptosis and multiple redox-related pathways based on KEGG annotation. Thus, we generated Ogt1-conditional knockout mice and found that Ogt1 deficiency aggravated ferroptosis, as evidenced by lipid reactive oxygen species (lipid ROS), malondialdehyde (MDA), Fe2+, as well as alterations in critical protein expression glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Consistently, we found that elevated O-GlcNAcylation inhibited ferroptosis sensitivity in hypoxia/reoxygenation (H/R) injury-induced TC-1 cells via O-GlcNAcylated NF-E2-related factor-2 (Nrf2). Furthermore, both the chromatin immunoprecipitation (ChIP) assay and the dual-luciferase reporter assay indicated that Nrf2 could bind with translation start site (TSS) of glucose-6-phosphate dehydrogenase (G6PDH) and promote its transcriptional activity. As an important rate-limiting enzyme in the pentose phosphate pathway (PPP), elevated G6PDH provided a mass of nicotinamide adenine dinucleotide phosphate (NADPH) to improve the redox state of glutathione (GSH) and eventually led to ferroptosis resistance. Rescue experiments proved that Nrf2 knockdown or Nrf2-T334A (O-GlcNAcylation site) mutation abolished the protective effect of ferroptosis resistance.ConclusionsIn summary, we revealed that O-GlcNAcylation could protect against I/R lung injury by reducing ferroptosis sensitivity via the Nrf2/G6PDH pathway. Our work will provide a new basis for clinical therapeutic strategies for pulmonary ischemia–reperfusion-induced acute lung injury.

High throughput single cell metagenomic sequencing with semi-permeable capsules: unraveling microbial diversity at the single-cell level in sewage and fecal microbiomes

Single-cell sequencing may serve as a powerful complementary technique to shotgun metagenomics to study microbiomes. This emerging technology allows the separation of complex microbial communities into individual bacterial cells, enabling high-throughput sequencing of genetic material from thousands of singular bacterial cells in parallel. Here, we validated the use of microfluidics and semi-permeable capsules (SPCs) technology (Atrandi) to isolate individual bacterial cells from sewage and pig fecal samples. Our method involves extracting and amplifying single bacterial DNA within individual SPCs, followed by combinatorial split-and-pool single-amplified genome (SAG) barcoding and short-read sequencing. We tested two different sequencing approaches with different numbers of SPCs from the same sample for each sequencing run. Using a deep sequencing approach, we detected 1,796 and 1,220 SAGs, of which 576 and 599 were used for further analysis from one sewage and one fecal sample, respectively. In shallow sequencing data, we aimed for 10-times more cells and detected 12,731 and 17,909 SAGs, of which we used 2,456 and 1,599 for further analysis for sewage and fecal samples, respectively. Additionally, we identified the top 10 antimicrobial resistance genes (ARGs) in both sewage and feces samples and linked them to their individual host bacterial species.

BRD4 Induces Esophageal Squamous Cell Carcinoma Progression via the Wnt/β-catenin Pathway.

BRD4, part of the bromodomain and extra terminal domain (BET) protein family, plays a pivotal role in gene transcription, DNA replication, and repair via transcription regulators. Despite its established involvement in various human diseases, its function in esophageal squamous cell carcinoma (ESCC) has not been fully explored. Our research investigated the association of BRD4 in ESCC and its underlying molecular mechanisms. The findings revealed that BRD4 knockdown notably diminished the cells' proliferation, migration, invasion capabilities and induced apoptosis and cell cycle arrest. Conversely, overexpression of BRD4 can reverse these phenotypes. Pearson correlation and enrichment analyses indicated that BRD4 expression was associated with the cell cycle and Wnt/β-catenin signaling pathway. Further validation confirmed that reduced BRD4 expression downregulates Cyclin D1 and c-Myc, and suppresses epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin signaling pathway. Furthermore, rescue experiments showed that overexpressing c-Myc significantly mitigated the inhibitory impact of BRD4. Moreover, by employing single-cell transcriptome sequencing, we explored the impact of the tumor microenvironment on BRD4 overexpression in ESCC cells. These insights confirmed BRD4's potential as a therapeutic target, suggesting that modulating its expression could yield promising strategies for ESCC treatment.

Efficient Identification of Monoclonal Antibodies Against Rift Valley Fever Virus Using High-Throughput Single Lymphocyte Transcriptomics of Immunized Mice

Background: Rift Valley fever virus (RVFV) is a zoonotic virus that poses a significant threat to both livestock and human health and has caused outbreaks in endemic regions. In humans, most patients experience a febrile illness; however, in some patients, RVF disease may result in hemorrhagic fever, retinitis, or encephalitis. While several veterinary vaccines are being utilized in endemic countries, currently, there are no licensed RVF vaccines or therapeutics for human use. Neutralizing antibodies specifically targeting vulnerable pathogen epitopes are promising candidates for prophylactic and therapeutic interventions. In the case of RVFV, the surface glycoproteins Gc and Gn, which harbor neutralizing epitopes, represent the primary targets for vaccine and neutralizing antibody development. Methods: We report the implementation of advanced 10x Genomics technology, enabling high-throughput single-cell analysis for the identification of rare and potent antibodies against RVFV. Following the immunization of mice with live attenuated rMP-12-GFP virus and successive Gc/Gn boosts, memory B cell populations (both general and antigen-specific) were sorted from splenocytes by flow cytometry. Deep sequencing of the antibody repertoire at a single-cell resolution, together with bioinformatic analyses, was applied for BCR pair selection based on their abundance and specificity. Results: Twenty-three recombinant monoclonal antibodies (mAbs) were selected and expressed, and their antigen-binding capacities were characterized. About half of them demonstrated specific binding to their cognate antigen with relatively high binding affinities. Conclusions: These antibodies could be used for the future development of efficacious therapeutics, as well as for studying virus-neutralizing mechanisms. The current study, in which the single-cell sequencing approach was implemented for the development of antibodies targeting the RVFV surface proteins Gc and Gn, demonstrates the effective applicability of this technique for antibody discovery purposes.

Phospho-seq: integrated, multi-modal profiling of intracellular protein dynamics in single cells.

Cell signaling plays a critical role in neurodevelopment, regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify cytoplasmic and nuclear proteins, including those with post-translational modifications, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines, induced pluripotent stem cells, and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories, shed light on gene regulatory relationships, and help explore the causes and effects of diverse cell signaling in neurodevelopment.

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes

Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genome assemblies revealed suggestive gene content differences and provided the opportunity for detailed genetic analyses. We created a single-cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined the spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting that derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how the novelties of these fish evolved.

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes.

Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genome assemblies revealed suggestive gene content differences and provided the opportunity for detailed genetic analyses. We created a single-cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined the spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting that derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how the novelties of these fish evolved.

Single-cell landscape of dynamic changes in CD8+ T cells, CD4+ T cells and exhausted T cells in hepatocellular carcinoma

Hepatocellular carcinoma has a high incidence and poor prognosis. In this study, we investigated the value of T-cell-related genes in prognosis by single-cell sequencing data in hepatocellular carcinoma. Twelve cases of hepatocellular carcinoma single-cell sequencing were included in the study. The high dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify gene modules associated with CD4+ T cells, CD8+ T cells and exhausted T cells. Altered signaling pathway activity in exhausted T cells was uncovered by the AUCell algorithm. xCELL, TIMER, QUANTISEQ, CIBERSORT and CIBERSORT-abs were performed to explore immune cell infiltration. Immune checkpoint inhibitor genes and TIDE methods were used to predict immunotherapy response. Finally, immunohistochemistry and real-time PCR were used to verify gene expression. The hdWGCNA algorithm identified 40 genes strongly associated with CD4+ T cells, CD8+ T cells and exhausted T cells. Seven genes were finally selected for transcriptome data modeling. The results of the three independent datasets suggested that the model had strong prognostic value. Model genes were critical factors influencing CD4+ T cell and CD8+ T cell infiltration in patients. The efficacy of PD-1 immunotherapy was higher in patients belonging to the low-risk group. Alterations in signaling pathways’ activity within exhausted T cells were crucial factors contributing to the decline in immune function. Differential expression of seven genes in CD8+ T cells, CD4+ T cells and exhausted T cells were key targets for improving immunotherapy response in HCC.

Mendelian randomization and multiomics comprehensively reveal the causal relationship and potential mechanism between atrial fibrillation and gastric cancer

ObjectiveGastric cancer is a harmful disease, the comorbidity mechanism and causality relationship between this disease and other diseases are worth studying.MethodsUsing a two-sample Mendelian Randomization method, this study revealed the potential causal effect of atrial fibrillation (AF) on gastric cancer (GC) risk by constructing a genetic instrument containing 136 AF associated SNPs. Subsequently, analysis identifies 62 AF-GC co-associated genes and constructs a protein-protein interaction network of key genes. High-throughput sequencing data were further used to analyze the association between the two and their impact on the survival outcome of gastric cancer.ResultsThe results showed that AF was negatively associated with gastric cancer, and further analysis revealed that this relationship was independent of GC risk factors such as chronic gastritis, Helicobacter pylori infection, and alcohol consumption. Enrichment analysis reveals associations of key genes with pathways related to cardiovascular disease, inflammatory gastrointestinal diseases, and tumorigenesis. Through single-cell sequencing data analysis, fibroblast subpopulations associated with the key gene set are identified in GC, showing significant correlations with cancer progression and inflammation regulation pathways. Transcription factor analysis and developmental trajectory analysis reveal the potential role of fibroblasts in GC development. Finally, prognosis analysis and gene mutation analysis using TCGA-STAD data indicate an adverse prognosis associated with the key gene set in GC.ConclusionThis study provides new insights into the association between AF and GC and offers novel clues for understanding its impact on the pathogenesis and therapeutic strategies of GC.

Unraveling the tumor microenvironment of esophageal squamous cell carcinoma through single-cell sequencing: A comprehensive review.

Esophageal squamous cell carcinoma (ESCC) is a highly heterogeneous and aggressive malignancy. The progression, invasiveness, and metastatic potential of ESCC are shaped by a multitude of cells within the tumor microenvironment (TME), including tumor cells, immune cells, endothelial cells, as well as fibroblasts and other cell types. Recent advancements in single-cell sequencing technologies have significantly enhanced our comprehension of the diverse landscape of ESCC. Single-cell multi-omics technology, particularly single-cell transcriptome sequencing, have shed light on the expression profiles of individual cells and the molecular characteristics of distinct tumor cell populations. This review summarizes the latest literature on single-cell research in the field of ESCC, aiming to elucidate the heterogeneity of tumor cells, immune cells, and stromal cells at the single-cell level. Furthermore, it explores the impact of cellular interactions within the TME on the progression of ESCC. By compiling a comprehensive overview of single-cell omics research on ESCC, this article aims to enhance our understanding of ESCC diagnosis and treatment by elucidating the intricate interplay within the TME. It explores the cellular composition, spatial arrangement, and functional attributes of the ESCC TME, offering potential therapeutic targets and biomarkers for personalized treatment strategies.

Single-cell transcriptome sequencing analysis of physiological and immune profiling of crucian carp (Carassius auratus) gills.

Gills are the main respiratory organs of fish and bear important physiological and immunological functions, but the functional heterogeneity of interlamellar cell mass (ILCM) at the single-cell level has rarely been reported. Here, we identified 19 cell types from the gills of crucian carp (Carassius auratus) by single-cell RNA sequencing (scRNA-seq) in combination with histological analysis. We annotated ILCM and analyzed its functional heterogeneity at the single-cell level for the first time. Functional enrichment analysis and cell cycle analysis identified ILCM as a type of metabolically active cells in a state of constant proliferation, and identified the major pathways responsible for ILCM immunoregulation. Histological analysis revealed the morphology and positional relationships of 6 cell types. Meanwhile, the gene regulatory network of ILCM was established through weighted gene co-expression network analysis (WGCNA), and one transcription factor and five hub genes related to immunoregulation were identified. We found that pyroptosis might be an important pathway responsible for the immune response of ILCM. Our findings provide an insight into the physiological and immune functions of gills and ILCM at the single-cell level and lay a solid foundation for further exploration of the molecular mechanism of ILCM immunity functions.

Cotton2035: From genomics research to optimized breeding.

Cotton is the world's most important natural fiber crop and serves as an ideal model for studying plant genome evolution, cell differentiation, elongation, and cell wall biosynthesis. The first draft genome assembly for Gossypium raimondii, completed in 2012, marked the beginning of global efforts in studying cotton genomics. Over the past decade, the cotton research community has continued to assemble and refine the genomes for both wild and cultivated Gossypium species. With the accumulation of de novo genome assemblies and resequencing data across virous cotton populations, significant progress has been made in uncovering the genetic basis of key agronomic traits. Achieving the goal of cotton genomics-to-breeding (G2B) will require a deeper understanding of the spatiotemporal regulatory mechanisms involved in genome information storage and expression. We advocate for a cotton ENCODE project to systematically decode the functional elements and regulatory networks within the cotton genome. Technological advances, particularly on single-cell sequencing and high-resolution spatiotemporal omics, will be essential for elucidating these regulatory mechanisms. By integrating multi-omics data, genome editing tools, and artificial intelligence, these efforts will empower the genomics-driven strategies needed for future cotton G2B breeding.

Mechanisms and strategies of immunosenescence effects on non-small cell lung cancer (NSCLC) treatment: A comprehensive analysis and future directions.

Non-small cell lung cancer (NSCLC), the most prevalent form of lung cancer, remains a leading cause of cancer-related mortality worldwide, particularly among elderly individuals. The phenomenon of immunosenescence, characterized by the progressive decline in immune cell functionality with aging, plays a pivotal role in NSCLC progression and contributes to the diminished efficacy of therapeutic interventions in older patients. Immunosenescence manifests through impaired immune surveillance, reduced cytotoxic responses, and increased chronic inflammation, collectively fostering a pro-tumorigenic microenvironment. This review provides a comprehensive analysis of the molecular, cellular, and genetic mechanisms of immunosenescence and its impact on immune surveillance and the tumor microenvironment (TME) in NSCLC. We explore how aging affects various immune cells, including T cells, B cells, NK cells, and macrophages, and how these changes compromise the immune system's ability to detect and eliminate tumor cells. Furthermore, we address the challenges posed by immunosenescence to current therapeutic strategies, particularly immunotherapy, which faces significant hurdles in elderly patients due to immune dysfunction. The review highlights emerging technologies, such as single-cell sequencing and CRISPR-Cas9, which offer new insights into immunosenescence and its potential as a therapeutic target. Finally, we outline future research directions, including strategies for rejuvenating the aging immune system and optimizing immunotherapy for older NSCLC patients, with the goal of improving treatment efficacy and survival outcomes. These efforts hold promise for the development of more effective, personalized therapies for elderly patients with NSCLC.

Comprehensive analysis reveals the tumor suppressor role of macrophage signature gene FCER1G in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) progression is closely linked to the role of macrophages. This study utilized single-cell RNA sequencing and genomic analysis to explore the characteristic genes of macrophages in HCC and their impact on patient prognosis. We obtained single-cell se-quencing data from seven HCC samples in the GEO database. Through principal component analysis and t-SNE dimensionality reduction, we identified 2,000 highly variable genes and per-formed clustering and annotation of 17 cell clusters, revealing 482 macrophage-related feature genes. A LASSO regression model based on these genes was developed to predict the prognosis of HCC patients, with validation in the TCGA-LIHC cohort demonstrating model accuracy (AUC = 0.78, 0.72, 0.71 for 1-, 3-, and 5-year survival rates, respectively). Additionally, patients in the high-risk group exhibited elevated tumor stemness scores, although no significant differences were observed in microsatellite instability (MSI) and tumor mutational burden (TMB) scores. Immune-related analyses revealed that FCER1G expression was downregulated in HCC and was associated with key pathways such as apoptosis and ferroptosis. Reduced FCER1G expression significantly affected HCC cell proliferation and migration. Our prognostic model provides new insights into precision and immunotherapy for HCC and holds significant implications for future clinical applications.

Exploring the therapeutic efficacy difference in claudin18.2-targeted cell therapy revealed by single-cell sequencing

Exploring the therapeutic efficacy difference in claudin18.2-targeted cell therapy revealed by single-cell sequencing