Single-cell RNA Sequencing Analysis Research Articles

Single-cell RNA sequencing of neonatal cortical astrocytes reveals versatile cell clusters during astrocyte-neuron conversion.

Astrocytes are extensively utilized as starting cells for neuronal conversion. Our previous study discovered that a portion of primary cultured mouse neonatal cortical astrocytes can be directly converted into neurons after exposure to a neurogenic induction condition. Recent in vivo studies have demonstrated astrocyte heterogeneity in terms of their developmental origin, molecular profile, physiology, and functional outputs. We hypothesized that the heterogeneity of primary astrocytes in our study could influence their conversion potential. We performed single-cell RNA sequencing on cells harvested at key time points during in vitro astrocyte-to-neuron conversion, specifically on Day 1 and Day 9. Through single-cell RNA sequencing analysis, we identified several subpopulations of astrocytes, labeled as Astrocyte 1 to Astrocyte 3, based on distinct gene expression patterns. Pseudotime trajectory analysis predicted the existence of three distinct cell states throughout the conversion process. Astrocyte 3 exhibited a higher propensity for neuronal conversion, with proliferation genes like Mki67 being highly expressed. Additionally, several candidate genes were identified as potentially crucial in the conversion process. Astrocyte 3 is considered a unique subtype population of astrocytes. Our investigation underscores the diversity of primary neonatal cortical astrocytes and provides critical insights into the potential for astrocyte-to-neuron conversion, which may be harnessed to enhance the efficiency of this astrocyte-neuron conversion process.

Abstract TP348: Overload of efferocytic cargoes in lysosome shifts microglia/macrophages towards pro-inflammatory phenotype through activating STING after stroke.

Objective: Incomplete clearance of cell debris in stroke lesion contributes to post-stroke cognitive impairment. Microglia and infiltrated macrophages are the major scavenger of debris in stroke lesion. However, as stroke progresses, their phagocytic capacity is impaired, while secretion of pro-inflammatory factors was enhanced. This study aims to identify the mechanisms driving the phenotypic shift after excessive phagocytosis. Methods: Brain cells from 8-12 week-old male C57BL/6J mice were collected for single-cell RNA sequencing at 1, 5, and 14 days after transient middle cerebral artery occlusion (tMCAO), as well as from sham-operated control group. Key transcription factors mediating the phenotypic changes of microglia and macrophages were identified through bioinformatics analysis, and further validated in vivo and in vitro using immunofluorescence staining, flow cytometry, and Western blot. Additionally, tMCAO models were treated with the key factor inhibitor to evaluate microglia and macrophage phenotypic shifts and the progression of stroke. Results: Through single-cell RNA sequencing analysis, we identified a concurrent activation pattern between the stimulator of interferon genes (STING) mediated Type I interferon signaling and the pro-inflammatory phenotype of microglia/macrophages. In the first 5 days after stroke, microglia and macrophages displayed an inflammatory-resolving phenotype with enhanced phagocytic activity, but from days 5 to 14, they transitioned to a pro-inflammatory phenotype with sustained upregulation of STING-mediated Type I interferon pathway molecules. We validated the phenomenon observed in bioinformatics using tMCAO models. In vitro and in vivo, we found that continuous phagocytosis by macrophages and microglia led to STING activation, triggering Type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. Treatment with STING inhibitor H-151 reversed these detrimental shifts.. Conclusions: These findings suggest that continuous post-stroke phagocytosis by macrophages and microglia led to the activation of STING, thereby triggering type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. STING inhibition may serve as a therapeutic strategy for mitigating neuroinflammatory responses following stroke.

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.

Targeting UBE2T suppresses breast cancer stemness through CBX6-mediated transcriptional repression of SOX2 and NANOG.

Breast cancer stem cells (BCSCs) are the main cause of breast cancer recurrence and metastasis. While the ubiquitin-proteasome system contributes to the regulation of BCSC stemness, the underlying mechanisms remain unclear. Here, we identified ubiquitin-conjugating enzyme E2T (UBE2T) as a pivotal ubiquitin enzyme regulating BCSC stemness through systemic screening assays, including single-cell RNA sequencing (scRNA-seq) and stemness-index analysis. We found that patients with high UBE2T expression exhibited worse prognosis than those with low expression (10-year PFS: 55.95% vs. 85.08%), which are consistent across various subtypes of breast cancers. Genetic ablation of UBE2T suppresses BCSC stemness and tumor progression in organoids and spontaneous MMTV-PyMT mice, dependent on the transcriptional inactivation of pluripotency genes SOX2 and NANOG. Mechanically, UBE2T collaborates with the E3 ligase TRIM25 to perform K48-linked polyubiquitination and degradation of CBX6 at K214, which deficiency helps to promote the transcription of SOX2 and NANOG and enhances BCSC stemness. The pharmacological inhibitor of UBE2T significantly reduced the expression of NANOG and SOX2, suppressed tumor progression, and demonstrated synergistic effects when combined with chemotherapeutics, but not with other treatments. Collectively, our study revealed that the UBE2T-TRIM25-CBX6 axis can regulate BCSC stemness and offers a potentially therapeutic strategy to combat breast cancer in a clinical translation setting.

Shared chemoresistance genes in ESCC and cervical Cancer: Insights from pharmacogenomics and Mendelian randomization.

Neoadjuvant chemotherapy, particularly the use of platinum-based compounds and taxanes, is pivotal in the treatment of epithelial-derived tumors, such as cervical cancer and esophageal squamous cell carcinoma (ESCC); however, resistance remains a significant challenge. Utilizing Mendelian randomization (MR) with pharmacogenomics offers a novel approach to understanding the genetic underpinnings of drug responses, thereby aiding in personalized treatment. Single-cell RNA sequencing (scRNA-seq) analysis revealed a shared cellular subpopulation of CD8+T effector memory (CD8+TEM) cells that are pivotal in mediating chemotherapy resistance in ESCC and cervical cancer. A two-sample approach was employed for MR using data from genome-wide association studies, focusing on single nucleotide polymorphisms (SNPs) linked to CD8+TEM cell expression. The SNPs were carefully selected, and statistical models, including the Wald ratio and inverse variance weighted methods, were used for robust causal effect estimation. These were supplemented by MR-Egger and weighted median analyses to address pleiotropy and variant heterogeneity. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and immunohistochemistry assays were used to verify the relationship between the gene and drug sensitivity. Increased proportion of CD8+TEM cells were observed in resistant samples. MR identified IL32, SPOCK1, and TRBC2 as key genes associated with resistance to cisplatin, carboplatin, and paclitaxel, respectively. These findings were validated across various cohorts and underscored the role of CD8+TEM cells in drug responsiveness. The results of the MTT and immunohistochemistry assays confirmed the MR findings. Our study highlights the significant role of CD8+TEM cells in the chemoresistance of ESCC and cervical cancer and identified three genetic markers crucial for resistance to common chemotherapeutic agents. These findings suggest potential pathways for developing personalized treatment strategies, offering clinically relevant insights that could enhance therapeutic efficacy and help overcome drug resistance in patients with ESCC or cervical cancer.

Single-cell RNA sequencing and functional analysis reveal the role of altered glycosylation levels of hepatic macrophages in liver cirrhosis.

Liver cirrhosis represents a critical stage of chronic liver disease, characterized by progressive liver damage, cellular dysfunction, and disrupted cell-to-cell interactions. Glycosylation, an essential post-translational modification, significantly influences cellular behavior and disease progression. Its role in cirrhosis at the single-cell level remains unclear, despite its importance. This study, based on single-cell glycosylation and transcriptome data, compared the expression of differentially expressed genes in liver tissues from cirrhotic and healthy control samples, identifying changes in glycosylation-related genes and their functional pathway enrichment characteristics. Additionally, it analyzed the composition of immune cells and intercellular interaction features, with a focus on the interaction between macrophages and other immune cells and their potential role in immune regulation. The analysis revealed significant changes in immune cell composition and glycosylation patterns in cirrhotic livers. Specifically, the number of macrophages increased substantially, while overall glycosylation levels decreased. Enhanced interactions between macrophages and other cell types were observed, highlighting the central role of macrophages in reshaping the immune microenvironment during cirrhosis progression. Gene expression analysis showed a marked upregulation of FUCA1, a gene encoding a glycosylation-related hydrolase. This change was strongly associated with the observed reduction in glycosylation levels. Functional enrichment analysis further revealed that glycosylation-related genes were primarily involved in immune pathways, including antigen processing and presentation, cytokine signaling, and immune activation. Single-cell glycosylation analysis provides crucial insights into immune cell interactions in cirrhosis. Targeting glycosylation pathways in macrophages may offer new treatment strategies for cirrhosis.

Retinoic acid antagonizes estrogen signaling to maintain adult uterine cell fate

Classical tissue recombination experiments demonstrate that cell-fate determination along the anterior-posterior axis of the Müllerian duct occurs prior to postnatal day 7 in mice. However, little is known about how these cell types are maintained in adults. In this study, we provide genetic evidence that a balance between antagonistic retinoic acid (RA) and estrogen signaling activity is required to maintain simple columnar cell fate in adult uterine epithelium. Transdifferentiation of simple columnar uterine epithelium into stratified cervicovaginal-like epithelium was observed in three related mouse genetic models, in which RA signaling was perturbed in the postnatal uterus. Single-cell RNA sequencing analysis identified the transformed epithelial cell populations and revealed extensive immune cell infiltration resulting from loss of RA signaling. Surprisingly, disruption of RA signaling led to dysregulated expression of a substantial number of estrogen target genes, suggesting that these two pathways may functionally oppose each other in determining and maintaining uterine epithelial cell fate. Consistent with this model, neonatal exposure to the strong synthetic estrogen, diethylstilbestrol, downregulated expression of a group of RA target genes and led to epithelial stratification and immune cell infiltration in wild-type uterus. Treating RA receptor triple conditional knockout pups with fulvestrant, an estrogen antagonist, reestablished the balance between the two signaling pathways, and effectively prevented the transformation of mutant simple columnar epithelia to metaplastic stratified epithelia. These findings implicate an essential role for RA signaling in maintaining uterine cytodifferentiation by antagonizing estrogen signaling in the postnatal uterus.

Cell signaling communication within papillary craniopharyngioma revealed by an integrated analysis of single-cell RNA sequencing and bulk RNA sequencing

ObjectiveThis study aims to elucidate the primary signaling communication among papillary craniopharyngioma (PCP) tumor cells.MethodsFive samples of PCP were utilized for single-cell RNA sequencing. The most relevant ligand and receptor interactions among different cells were calculated using the CellChat package in R software. Bulk RNA sequencing of 11 tumor samples and five normal controls was used to investigate the pair interactions detected by single-cell RNA sequencing.ResultsFibroblasts were not found in ACP, whereas they were detected in PCP. InferCNV revealed high CNV scores for the clusters of epithelial cells and fibroblasts using immune cells as a reference. Epithelial Mesenchymal Transition, Interferon Gamma Response, p53 Pathway, and Estrogen Response Early are pathways commonly shared by fibroblasts and epithelial cells, ranking high in priority. The Wnt signaling pathway and PI3K-Akt signaling pathway play a crucial role in facilitating communication between epithelial cells and fibroblasts. Neutrophils were recognized as the main receivers of incoming signals, with ANXA1-FPR1 and MIF-(CD74 + CXCR2) being identified as the primary signals transmitted from fibroblasts to neutrophils.ConclusionThrough analyzing the communication of essential signaling pathways, ligands, and receptors among epithelial cells, fibroblasts, and neutrophils in PCP tumor tissues, we have identified certain molecules with promising prognostic and therapeutic potential.

Intestinal Bacteroides drives glioma progression by regulating CD8+ T cell tumor infiltration.

The intestinal microbiota regulates normal brain physiology and the pathogenesis of several neurological disorders. While prior studies suggested that this regulation operates through immune cells, the underlying mechanisms remain unclear. Leveraging two well characterized murine models of low-grade glioma (LGG) occurring in the setting of the neurofibromatosis type 1 (NF1) cancer predisposition syndrome, we sought to determine the impact of the gut microbiome on optic glioma progression. Nf1-mutant mice genetically engineered to develop optic pathway gliomas (Nf1OPG mice) by 3 months of age were reared under germ-free (GF) conditions, treated with specific cocktails of antibiotics, or given fecal matter transplants (FMTs). Intestinal microbial species were identified by 16S genotyping. Neutralizing TGFβ antibodies were delivered systemically, while in vitro experiments used isolated murine microglia and T cells. Single cell RNA sequencing analysis was performed using established methods. Nf1 OPG mice raised in a GF environment or postnatally treated with vancomycin did not harbor optic gliomas or exhibit OPG-induced retinal nerve fiber layer thinning, which was reversed following conventionally raised mouse FMT or colonization with Bacteroides species. Moreover, this intestinal microbiota-regulated gliomagenesis was mediated by circulating TGFβ, such that systemic TGFβ neutralization reduced Nf1-OPG growth. TGFβ was shown to act on tumor-associated monocytes to induce Ccl3 expression and recruit CD8+ T cells necessary for glioma growth. Taken together, these findings establish, for the first time, a mechanistic relationship between Bacteroides in the intestinal microbiome and NF1-LGG pathobiology, suggesting both future predictive risk assessment strategies and therapeutic opportunities.

Single-cell analyses of intestinal epithelium reveal the dysregulation of gut immune microenvironment in systemic lupus erythematosus

BackgroundThe small intestine harbors a rich array of intestinal intraepithelial lymphocytes (IELs) that interact with structural cells to collectively sustain gut immune homeostasis. Dysregulation of gut immune homeostasis was implicated in the pathogenesis of multiple autoimmune diseases, however, whether this homeostasis is disrupted in a lupus autoimmune background remains unclear.MethodsWe performed single-cell RNA sequencing (scRNA-seq) analyses to elucidate immune and structural milieu in the intestinal epithelium of MRL/Lpr lupus mice (Lpr mice) and MRL/Mpj control mice (Mpj mice). Comprehensive analyses including unsupervised clustering, trajectories, and cellular communication were performed. The primary findings from scRNA-seq were further validated by quantitative polymerase chain reaction (qPCR), flow cytometry, and in vivo experiments including selenium supplementation.ResultsWe observed a significant reduction in CD8αα + IELs, accompanied by a marked increase in CD8αβ + IELs in Lpr mice. Additionally, subsets of CD8 + IELs exhibiting significantly enhanced effector functions were found to be markedly enriched in Lpr mice. Intercellular communication patterns within intestinal epithelial immune and structural cells were found to be specifically altered in Lpr mice. Moreover, scRNA-seq revealed significantly decreased intestinal TCRγδ T cells (γδT) associated with reduced aryl-hydrocarbon receptor repressor (AHRR) expression and subsequent oxidative stress and ferroptosis in Lpr mice. Antioxidant selenium effectively reversed the loss of γδT in Lpr mice, improved the gut barrier, and alleviated lupus symptoms.ConclusionsOur high-resolution single-cell atlas enhances the understanding of the immune and structural milieu of intestinal epithelium in lupus and provides new insights into lupus pathogenesis mediated by intestinal immune dysregulation.

Wnt signalling facilitates neuronal differentiation of cochlear Frizzled10-positive cells in mouse cochlea via glypican 6 modulation

Degeneration of cochlear spiral ganglion neurons (SGNs) leads to irreversible sensorineural hearing loss (SNHL), as SGNs lack regenerative capacity. Although cochlear glial cells (GCs) have some neuronal differentiation potential, their specific identities remain unclear. This study identifies a distinct subpopulation, Frizzled10 positive (FZD10+) cells, as an important type of GC responsible for neuronal differentiation in mouse cochlea. FZD10 + cells can differentiate into various SGN subtypes in vivo, adhering to natural proportions. Wnt signaling enhances the ability of FZD10 + cells to function as neural progenitors and increases the neuronal excitability of the FZD10–derived neurons. Single-cell RNA sequencing analysis characterizes FZD10-derived differentiating cell populations, while crosstalk network analysis identifies multiple signaling pathways and target genes influenced by Wnt signaling that contribute to the function of FZD10 + cells as neural progenitors. Pseudotime analysis maps the differentiation trajectory from proliferated GCs to differentiating neurons. Further experiments indicate that glypican 6 (GPC6) may regulate this neuronal lineage, while GPC6 deficiency diminishes the effects of Wnt signaling on FZD10–derived neuronal differentiation and synapse formation. These findings suggest the critical role of Wnt signaling in the neuronal differentiation derived from cochlear FZD10 + cells and provide insights into the mechanisms potentially involved in this process.

Genetic variation in IL-4 activated tissue resident macrophages determines strain-specific synergistic responses to LPS epigenetically

How macrophages in the tissue environment integrate multiple stimuli depends on the genetic background of the host, but this is still poorly understood. We investigate IL-4 activation of male C57BL/6 and BALB/c strain specific in vivo tissue-resident macrophages (TRMs) from the peritoneal cavity. C57BL/6 TRMs are more transcriptionally responsive to IL-4 stimulation, with induced genes associated with more super enhancers, induced enhancers, and topologically associating domains (TAD) boundaries. IL-4-directed epigenomic remodeling reveals C57BL/6 specific enrichment of NF-κB, IRF, and STAT motifs. Additionally, IL-4-activated C57BL/6 TRMs demonstrate an augmented synergistic response upon in vitro lipopolysaccharide (LPS) exposure, despite naïve BALB/c TRMs displaying a more robust transcriptional response to LPS. Single-cell RNA sequencing (scRNA-seq) analysis of mixed bone marrow chimeras indicates that transcriptional differences and synergy are cell intrinsic within the same tissue environment. Hence, genetic variation alters IL-4-induced cell intrinsic epigenetic reprogramming resulting in strain specific synergistic responses to LPS exposure.

CD271high cancer stem cells regulate macrophage polarization in head and neck squamous cell carcinoma.

Cancer stem cells (CSCs) are considered key drivers of progression in head and neck squamous cell carcinoma (HNSCC). Our single-cell RNA sequencing (scRNA-seq) analysis revealed predominant expression of CD271 in CSCs, however, its role as a CSC marker in HNSCC requires further elucidation. We investigated the stemness characteristics of CD271high HNSCC cells and their interactions with the tumor immune microenvironment. scRNA-seq data from hypopharyngeal squamous cell carcinoma (HPSCC) tissues were analyzed to identify expression profile of CSCs. Overall survival was compared between CD271high and CD271low patients based on immunostaining of HPSCC samples. The stemness of CD271high HNSCC cells was evaluated via an in vivo limiting dilution assay. In a C57BL/6 mice model, the percentage of immune cells and macrophage subtypes were analyzed by flow cytometry. The role of CD271 in macrophage polarization was further examined by in vitro coculture of CD271high cells with CD14+ monocytes. Gene expressions were analyzed by qPCR. CD271 is predominantly expressed in CSCs identified by scRNA-seq analysis. CD271 enhances HNSCC cell proliferation and is negatively correlated with patient prognosis in HPSCC. CD271 knockdown suppressed HNSCC tumor growth and regulated macrophage polarization within the TME. CD271high cells exhibited stemness features and enhanced tumor growth in vivo. CD271high HNSCC cells exhibit CSC characteristics and regulate macrophage polarization. Targeting CD271 may improve the immunosuppressive TME to inhibit tumor growth. Combining CD271-targeting agents with other therapies presents a promising strategy that may enhance therapeutic efficacy and prognosis in HNSCC.

P0827 Single cell RNA sequencing analysis of specific cell subsets for predicting anti-TNF treatment response in Korean patients with ulcerative colitis

Abstract Background The disruption of the epithelial barrier and mucosal homeostasis is key in developing ulcerative colitis (UC). Recent advances in single-cell RNA sequencing (scRNA-seq) have shed light on gene expression and cell characteristics in the intestine. However, there is limited research on Asian UC patients receiving anti-tumor necrosis factor (anti-TNF) treatment. This study sought to identify relevant cellular subsets in Korean UC patients after anti-TNF therapy using scRNA-seq. Methods This prospective study was conducted at two university hospitals in South Korea. To identify inflammatory cells that contribute to UC and are influenced by anti-TNF treatment, we performed scRNA-seq on sigmoid colonic biopsies from two healthy controls and six biologic-naïve UC patients prior to anti-TNF therapy. Mucosal biopsies from patients with active moderate to severe UC were collected at baseline (n=6) and again at a 52-week follow-up after anti-TNF treatment (n=2; sample collected form the responder), at which point treatment response was evaluated. A treatment responder was defined as a patient who maintained clinical remission (partial Mayo score of ≤2, with no subscore >1 and a rectal bleeding subscore of 0) at 52 weeks. Results After processing the scRNA-seq data, 13 clusters were identified, and cell clusters were annotated using left colon marker genes (Figure 1-a). Among the six patients evaluated at 52 weeks after anti-TNF treatment, five were treatment responders and one was a non-responder. Lower populations of ILC2 and ILC3 (Type 2 and Type 3 innate lymphoid), as well as CD8 T cells, CD4 T cells, and NK cells, were observed in the non-responder group compared to the responder group. Additionally, higher populations of mast cells, stromal fibroblasts, and endothelial cells were found in the non-responder group (Figure 1-b). Conclusion In Korean UC patients, anti-TNF treatment is linked to changes in tissue ILC2 and ILC3 subsets. scRNA seq analysis of tissue specimens obtained before anti-TNF use reveals stromal fibroblasts, endothelial, and mast cell-enriched genes related to anti-TNF responsiveness, underscoring the significant roles of these subsets in UC. Further confirmation is needed with larger samples and clinical validation

P0086 The role of enteric glial cells in patients with ulcerative colitis.

Abstract Background Enteric glial cells (EGCs) have traditionally been regarded as supportive cells within the enteric nervous system. However, recent studies indicate that, beyond their involvement in neural circuit formation, EGCs possess antigen-presenting capabilities and actively contribute to mucosal defense. In the mouse myenteric plexus, inflammation-inducing stimuli have been shown to trigger the expression of MHC class II on glial cells, which subsequently influences the differentiation of helper T-cell and regulatory T-cell subtypes, thereby maintaining immune homeostasis under inflammatory conditions. Nevertheless, research on EGCs in humans remains limited. Thus, this study aimed to elucidate the function and involvement of EGCs in the pathogenesis of ulcerative colitis (UC). Methods Normal colonic mucosa was obtained from histologically and macroscopically intact areas of patients with colorectal cancer (n=6). Colonic mucosa from UC patients, diagnosed based on established clinical, bacteriological, radiologic, and endoscopic criteria, was also obtained from surgically resected specimens (n=7). Lamina propria mononuclear cells were isolated through enzymatic digestion, and mesenchymal stromal fractions (CD31-CD45-EPCAM-CD90+), containing glial cells, were further isolated using flow cytometry for single-cell RNA sequencing (scRNA-seq) analysis and T cell differentiation assay. Results We identified a specific EGC population (Cluster 3: PDPN+ HLA-DR high) with high antigen-presenting capacity, which was selectively elevated at inflamed sites in UC. scRNA-seq analysis, transcription factor activity analysis, and gene-enriched pathway analysis indicated that IFNγ-STAT1 signaling activation within Cluster 3 enhances MHC class II expression. To assess T-cell effects, naive CD4+ T-cells were co-cultured with PDPN+ HLA-DR high cells isolated from UC samples. This co-culture induced differentiation into IL-10+ IL-17A+ CD4+ T-cells, with a positive correlation observed between the proportions of IL-10+ IL-17A+ CD4+ T-cells and PDPN+ HLA-DR high cells. Bulk RNA sequencing and qPCR analysis further demonstrated that PDPN+ HLA-DR high cells highly express the IL-27 subunit EBI3 and the TGF-β activator integrin αvβ8. To evaluate clinical relevance, we also examined the correlation between PDPN+ HLA-DR high cell numbers and clinical factors in 45 UC patients. Immunofluorescence staining for the glial marker CDH2 and HLA-DR revealed a negative correlation between CDH2+ HLA-DR+ cell numbers and preoperative Mayo endoscopic scores. Conclusion PDPN+ HLA-DR high glial cells are increased in inflamed areas of UC patients, suggesting a role in anti-inflammatory processes and the maintenance of immune homeostasis.

Comprehensive single-cell atlas of colorectal neuroendocrine tumors with liver metastases: unraveling tumor microenvironment heterogeneity between primary lesions and metastases

BackgroundColorectal neuroendocrine tumors with liver metastases (CRNELM) are associated with a poorer prognosis compared to their nonmetastatic counterparts. A comprehensive understanding of the tumor microenvironment (TME) heterogeneity between primary lesions (PL) and liver metastases (LM) could provide crucial insights for enhancing clinical management strategies for these patients.MethodsWe utilized single-cell RNA sequencing to analyze fresh tissue samples from CRNELM patients, aiming to elucidate the variations in TME between PL and LM. Complementary multidimensional validation was achieved through spatial transcriptomics, bulk RNA sequencing, and multiplex immunohistochemistry/immunofluorescence.ResultsOur single-cell RNA sequencing analysis revealed that LM harboured a higher proportion of CD8 + T cells, CD4 + T cells, NK cells, NKT cells, and B cells exhibiting a stress-like phenotype compared to PL. RGS5 + pericytes may play a role in the stress-like phenotype observed in immune cells within LM. MCs in PL (PL_MCs) and LM (LM_MCs) exhibit distinct activation of tumor-associated signaling pathways. Notably, COLEC11 + matrix cancer-associated fibroblasts (COLEC11_mCAFs) were found to be significantly associated with LM_MCs. Cell communication analysis unveiled potential targetable receptor-ligand interactions between COLEC11_mCAFs and LM_MCs. Multidimensional validation confirmed the prominence of the characteristic stress-like phenotypes, including HSPA6_CD8_Tstr, HSPA6_NK, and COLEC11_mCAFs in LM. Moreover, a higher abundance of COLEC11_mCAFs correlated with poorer survival rates in the neuroendocrine tumor patient cohort.ConclusionOverall, our study provides the first single-cell analysis of the cellular and molecular differences between PL and LM in CRNELM patients. We identified distinct cell subsets and receptor-ligand interactions that may drive TME discrepancies and support metastatic tumor growth. These insights highlight potential therapeutic targets and inform strategies for better managing CRNELM patients.

ScRICA: An R package for multiple-sample single-cell RNA-seq data integrative comparative analysis

Single-cell sequencing technologies offer unprecedented resolution to inspect transcriptomes and generate critical biological insights. As the number of cells and cell types increase in single-cell studies, the effort required to analyze the data surges dramatically, especially when comparative explorations need to be performed on large datasets with different cell types and various sample attributes, such as clinical samples from different age and ancestry groups. Due to the sequential nature of single-cell data analysis, many steps involving multiple method choices and parameter options need to be considered. The computational skills required for integrative and comparative analyses of large datasets with various sample attributes represent a substantial obstacle for many researchers. To address this challenge, we have developed scRICA, a systematic workflow tailored for integrative and comparative single-cell RNA sequencing (scRNA-seq) analysis. This approach streamlines the analytical process, ensuring efficient utilization of computational resources and facilitating scalability for large-scale datasets. With scRICA, researchers can conduct integrative and comparative scRNA-seq analyses with ease, empowering them to derive meaningful insights from their data in a timely manner. scRICA offers a versatile approach by allowing users to input various parameter options from a metadata table, which are inherited throughout the entire analysis workflow. This functionality greatly enhances the efficiency of programming for comparative analyses involving multiple sample attributes. As an R package, scRICA provides a user-friendly interface within the R environment, making it accessible to researchers familiar with R programming. Additionally, scRICA offers a command line execution option, allowing users to seamlessly integrate it into their computational pipelines or execute analyses on High-Performance Computing (HPC) systems. This combination of features ensures flexibility, ease of use, and scalability, making scRICA a valuable tool for comprehensive and efficient single-cell RNA sequencing analysis.

Polycystin-1 regulates tendon-derived mesenchymal stem cells fate and matrix organization in heterotopic ossification

Mechanical stress modulates bone formation and organization of the extracellular matrix (ECM), the interaction of which affects heterotopic ossification (HO). However, the mechanically sensitive cell populations in HO and the underlying mechanism remain elusive. Here, we show that the mechanical protein Polysyctin-1 (PC1, Pkd1) regulates CTSK lineage tendon-derived mesenchymal stem cell (TDMSC) fate and ECM organization, thus affecting HO progression. First, we revealed that CTSK lineage TDMSCs are the major source of osteoblasts and fibroblasts in HO and are responsive to mechanical cues via single-cell RNA sequencing analysis and experiments with a lineage tracing mouse model. Moreover, we showed that PC1 mediates the mechanosignal transduction of CTSK lineage TDMSCs to regulate osteogenic and fibrogenic differentiation and alters the ECM architecture by facilitating TAZ nuclear translocation. Conditional gene depletion of Pkd1 or Taz in CTSK lineage cells and pharmaceutical intervention in the PC1-TAZ axis disrupt osteogenesis, fibrogenesis and ECM organization, and consequently attenuate HO progression. These findings suggest that mechanically sensitive CTSK-lineage TDMSCs contribute to heterotopic ossification through PC1-TAZ signaling axis mediated cell fate determination and ECM organization.

Integrative single-cell and multi-omics analyses reveal ferroptosis-associated gene expression and immune microenvironment heterogeneity in gastric cancer

Gastric cancer (GC), a prevalent malignancy worldwide, encompasses a multitude of biological processes in its progression. Recently, ferroptosis, a novel mode of cell demise, has become a focal point in cancer research. The microenvironment of gastric cancer is composed of diverse cell populations, yet the specific gene expression profiles and their association with ferroptosis are not well understood. Our study employed single-cell RNA sequencing to thoroughly investigate the transcriptomic profiles and identify differential gene expression in gastric cancer, offering fresh insights into the cellular diversity and underlying molecular mechanisms of this disease. We discovered a set of significantly differentially expressed genes in GC, which may serve as valuable leads for future functional investigations. Subsequent analyses, including gene set intersection and functional enrichment, pinpointed genes implicated in ferroptosis and conducted comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to elucidate their biological roles. In the gene selection and model validation section, critical genes were identified using machine learning algorithms, constructing a model with high predictive accuracy. Besides, distorted immune landscapes were further identified in RBL using ssGSEA analysis such that the complex association of gene expression features and its interaction networks as well as infiltration by various types of immune cells can be more clearly understood. Correlation analysis with different immune cell subtypes showed CTSB as an important regulator in the distributions of cancer infiltrating cells. Single-cell RNA sequencing analysis was utilized to map the cellular composition and gene expression profiles of cells in the gastric cancer microenvironment, which provide critical information for elucidating cellular heterogeneity as well as tumor microenvironment regulation in GC. Moreover, the distribution of FTH1, ZFP36 and CIRBP at different expression levels show new research prospects for functional information of these promoters in tumor microenvironment. In summary, the present study augments our knowledge of molecular mechanisms underlying gastric tumorigenesisa and provide scientific basis for identifing new targets and biomarkers in therapeutic diagnosis.

Derivation of a Human Brain Organoid with Microglia Development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro toolfor studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporatedinto iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.