Single-cell Sequencing Research Articles

EPCO-51. A MULTI-OMIC ANALYSIS OF REGIONAL HETEROGENEITY IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults. A hallmark of GBM is its intratumoral heterogeneity as well as infiltration into the surrounding brain. The growing understanding of the cellular diversity and cellular state diversity within GBM necessitates a need for a more granular evaluation of the molecular landscape of this disease. This study aimed to investigate this using single cell RNA sequencing combined with single-cell ATAC sequencing and spatial transcriptomics in order to delineate cellular states and enriched pathways between malignant cells in different regions of GBM. The study cohort consisted of 15 patients with primary GBM. Tumor samples were taken at the time of surgical resection using intraoperative stereotactic navigation from three anatomically distinct locations: periphery – the cortex or white matter in the peri-tumoral region that is beyond contrast-enhancing tumor, border – the contrast-enhancing border of the tumor on T1-weighted MRI, and core - the hypointense core region on T1-weighted MRI. Samples underwent combined snRNA and ATAC sequencing (10x Multiome) as well as spatial transcriptomics (10x Visium). Single-nucleus RNA + ATAC sequencing captured a total of 37,547 neoplastic cells and 18,498 non-neoplastic cells within the tumor microenvironment. The majority of non-neoplastic cells were identified within the periphery alongside a distinct group of neoplastic cells that exhibited unique a unique distribution of malignant cellular state and differentially regulated genetic pathways compared to malignant cells within the tumor border and core. Gene regulatory analysis of the malignant cells in the periphery identified a previously undefined set of enriched transcription factors that are periphery-specific regulon drivers. Overall, this analysis sheds light on the genetic and epigenetic differences between malignant GBM cells based on their location and warrant further investigation into the biology and targetable aspects of the malignant cells that exist beyond the contrast-enhancing border of the tumor.

EPCO-19. MULTIOMIC PROFILING OF EPIGENETIC HETEROGENEITY IN GLIOBLASTOMA AT SINGLE-CELL RESOLUTION USING MULTIOMIC SINGLE-CELL CUT&TAG

Abstract INTRODUCTION Recent evidence suggests epigenetic heterogeneity may be the primary driver of cell phenotype and treatment resistance in glioblastoma (GBM). Epigenetic heterogeneity is defined by alterations of chromatin histone modifications with activating marks such as H3K27 acetylation and repressive marks such as H3K27 trimethylation. Combinations of these histone marks govern the activity of functional genomic elements such as enhancers or promoters. Single-cell (“sc-”) technologies have only recently been adapted to the study of epigenetic cell states and significant technical challenges have limited their widespread adoption, especially in complex tissues. Here, we report the development of an optimized method for performing tissue processing and simultaneous single-cell epigenetic and transcriptomic analyses in flash frozen GBM samples. METHODS Building on our prior work, we developed a new protocol, multiomic single-cell cleavage-under-targets and tagmentation, or “mscCUT&TAG”. We optimized nuclei isolation, cleanup and permeabilization from flash frozen GBM and reaction conditions for the scCUT&TAG portion of the assay. Critically, these changes maintained high-quality, intact nuclei in suspension while limiting well-described technical issues such as nuclei clumping and sample loss. Furthermore, we incorporated the ability to multiplex both patient samples and histone marks. RESULTS We applied 10X scRNA-seq, 10X Multiome (RNA+ATAC-seq), and mscCUT&TAG for five histone marks to the same GBM sample, enabling an integrated evaluation of the tumor’s transcriptome, chromatin accessibility and epigenetic regulation at single-cell resolution. mscCUT&TAG enabled the first assessment of single-cell genome segmentation in GBM. We integrated across modalities to identify specific tumor cell populations, cell-type specific coordinated changes in epigenetics and transcriptome, and define functional regulatory elements. CONCLUSIONS We developed a novel multiomic protocol, mscCUT&TAG, that allows for a comprehensive analysis of epigenetic heterogeneity in GBM. We anticipate this protocol will enhance our understanding of fundamental biology and potential therapeutic avenues in GBM.

EPCO-57. INTEGRATED ANALYSIS OF BULK AND SINGLE-CELL RNA SEQUENCING DATA OF PRIMARY AND METASTATIC PEDIATRIC MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a highly aggressive and the most common brain tumor in childhood. MB presents a high intertumoral heterogeneity, with at least four molecular subgroups identified (SHH, WNT, Group 3, and Group 4). Metastatic MB, or Leptomeningeal Disease (LMD), is predominantly found in the MB Group 3 type. Although LMD represents a main clinical challenge, its molecular mechanisms remain poorly characterized. Recent research has shown that primary and MB metastasis diverge dramatically. Our work has focused on establishing therapy naïve Group 3 Patient-Derived Xenografts models of primary and metastatic Medulloblastoma to conduct transcriptomic profiling at the bulk and single-nuclei RNAseq levels to identify genetic drivers/pathways that sustain leptomeningeal disease compartment. Our results show various signaling pathways enriched across LMD models, such as MYC targets, unfolded protein response, and fatty acid metabolism. Using single-sample GSEA analysis (ssGSEA) and deconvolution approaches, we have also identified that our PDXes models retain neoplastic subpopulations previously identified in MB single-cell sequencing studies. Similarly, we have identified slight differences in cell subpopulation proportions between primary and leptomeningeal compartments. Our single-nuclei studies have confirmed these results and differentially expressed genes previously found in bulk RNAseq analyses. These results suggest the presence of cell populations enriched in the metastatic compartment with an aberrant transcription phenotype and adaptations in fatty acid metabolism to survive the leptomeningeal space. In conclusion, our work supports the notion that primary and LMD retain subpopulation clusters present in MB Group 3 tumors with different proportions in the metastatic compartment shown by bulkRNAseq deconvolution and single nuclei RNA analysis. We also show that primary and LMD are transcriptionally different, with various enriched pathways in the metastatic compartment and shared among LMD Group 3 PDX models. Through these approaches, we aim to elucidate the genetic dependencies of metastatic Medulloblastoma that will help for targeted therapies.

EXTH-11. PEDIATRIC GLIOMA IMMUNE PROFILING IDENTIFIES TIM3 AS A THERAPEUTIC TARGET IN BRAF-FUSION PILOCYTIC ASTROCYTOMA

Abstract Despite being the leading cause of childhood mortality, pediatric gliomas have been relatively understudied. Repurposing of adult brain tumor immunotherapies in pediatric patients has not been successful, highlighting the need for pediatric-specific immunotherapies. Pilocytic astrocytomas (PA) are the most common pediatric glioma. While surgical resection of PA is the first-line treatment, the ten-year progression-free survival rate for patients with residual tumors is <50%. Notably, some PAs undergo spontaneous regression after partial resection, suggesting potential baseline tumor immunoreactivity. Whole transcriptome sequencing performed on a commercial platform of pediatric gliomas (n=250) showed that MAPK-driven gliomas have higher interferon signatures compared to other molecular subgroups. Single-cell sequencing identified a unique activated and cytotoxic microglia population designated MG-Act in BRAF-fused MAPK-activated PA (n=13; p < 0.05), but not in pediatric high-grade gliomas (n=5) or normal brain (n=3). TIM3 was expressed on the luminal side of endothelial cells and MG-Act, but not in the surrounding normal brain based on sequential multiplex imaging. Flow cytometry showed that TIM3 intensity is upregulated on immune cells in the PA tumor microenvironment (TME) relative to matched peripheral blood immune cells (n=6; p < 0.01). Anti-TIM3 reprogrammed fresh ex vivo immune cells from the TME of human PAs (n=3) to a pro-inflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven low-grade gliomas, median survival (MS) was 252 days in mice treated with anti-TIM3 (n=21; p<0.01) relative to IgG control (MS: 110 days; n=20) or anti-PD-1 (MS: 134 days; n=20). The therapeutic activity of anti-TIM3 was abrogated in the CX3CR1 microglia knockout background. ScRNA sequencing data during the therapeutic window of anti-TIM3 in wild-type mice demonstrated enrichment of the MG-Act population within low-grade gliomas at two different longitudinal time points but not in IgG-treated controls. Together, these data indicate that anti-TIM3 should be considered for clinical trials in pediatric low-grade MAPK-driven gliomas.

Feedback regulation of VISTA and Treg by TNF-α controls T cell responses in drug allergy.

Severe cutaneous adverse reactions (SCARs) mediated by cytotoxic T lymphocytes are a series of life-threatening conditions with a mortality of 4%-20%. The clinical application of tumor necrosis factor-alpha (TNF-α) antagonist improves the outcome of some SCARs patients; however, this is complicated by the elusive and varied immunopathogenesis. To investigate whether IgE antibody responses to HEMAs are associated with AD, its severity, and response to dupilumab. To clarify the precise process and optimize the therapy regimen of SCARs, we performed single-cell sequencing, invitro functional and clinical analysis of patients with SCARs. We observed that TNF-α breaks drug-specific T-cell tolerance by inhibiting the expression of V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA). Furthermore, TNF-α generated a positive feedback loop in the early phase of drug-specific T-cell activation, whereby B cells acted reciprocally on the corresponding T cells to reinforce TNF-α cytokine expression. In contrast, this pathway of TNF-α-VISTA signaling did not operate in memory effector T cells. Drug-specific memory effector T-cell responses were inhibited by increasing Treg cell expression in a negative feedback loop, with TNF-α antagonists preventing the inhibitory effect. These observations align with the clinical analysis that early but not late intervention with TNF-α antagonists significantly improved outcomes in SCARs patients. Our findings defining feedback regulation of VISTA and Treg cells by TNF-α in different stages of the drug-specific T-cell response and, indicate that a Treg agonists, instead of TNF-α antagonists, could be used for treatment of patients with progressive SCARs.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

EPCO-28. UNVEILING INVASIVE MECHANISMS OF GLIOBLASTOMA CELLS THROUGH MULTIMODAL SINGLE-CELL SEQUENCING

Abstract Glioblastoma (GBM) is the most common malignancy of the central nervous system, characterized by a dismal prognosis and inevitable recurrence despite aggressive standard-of-care therapy. Extensive colonization of the surrounding brain parenchyma precludes complete surgical resection, posing a significant therapeutic challenge. While well-studied in model systems, the molecular features and epigenetic regulators of invasive GBM cells remain under-explored directly in clinical samples. To address this gap, we performed a multimodal single-cell sequencing characterization of GBM tumor samples from anatomically distinct regions collected using MRI guidance. Our results demonstrate an enrichment of progenitor-like (neural- and oligodendrocyte-like) malignant states and neurons at the tumor margins. In contrast, differentiated-like states and myeloid cells were more abundant in the tumor core. Peri-tumoral progenitor-like malignant states expressed a unique neuronal signature associated with synaptic signaling, neurogenesis, and Notch signaling. Further, the expression of this neuronal signature was correlated with increased invasiveness. Analysis of matched primary-recurrent GBM patient cohorts revealed an expansion of this neuronal activity program, which was associated with worse overall survival. Motifs of proneural transcription factors implicated in neuronal lineage differentiation were also found to be differentially accessible in progenitor-like malignant states marked by the neuronal invasive signature, potentially contributing to the remodeling of cell states at the invasive margin. Further, cell-cell interaction analysis predicted greater communication between neurons and peri-tumoral progenitor-like malignant states, mediated predominantly through neurexin-neuroligin trans-synaptic signaling, facilitating synaptogenesis and the integration of tumor cells into neural circuits. Altogether, these findings suggest a model in which GBM invasive cells communicate with normal brain neurons in peri-tumoral regions, exploiting neurodevelopmental pathways to facilitate invasion and potentially seed recurrence. Our characterization of invasive GBM cells provides insight into the mechanisms of brain invasion and highlights potential therapeutic vulnerabilities of malignant invasive cells. Understanding these mechanisms opens new avenues for targeted therapies aimed at curtailing the invasive and adaptive capabilities of GBM.

CAF-macrophage crosstalk in tumour microenvironments governs the response to immune checkpoint blockade in gastric cancer peritoneal metastases

BackgroundPeritoneal metastasis is the most common metastasis pattern of gastric cancer. Patients with gastric cancer peritoneal metastasis (GCPM) have a poor prognosis and respond poorly to conventional treatments. Recently, immune...

Single cell analysis identified a basal cell transition state associated with the development and progression of bladder cancer

BackgroundBladder cancer (BC) is a prevalent malignancy characterized by significant cellular heterogeneity. While single-cell multi-omics studies have provided valuable insights, much of the existing data remains underexplored, limiting our understanding of BC’s molecular mechanisms. Uncovering the pathogenesis of BC and finding new treatment methods are urgent problems to be solved. This study aims to address this gap by re-analyzing available single-cell datasets to uncover novel insights into BC.MethodsIn this study, we retrieved three single-cell transcriptome datasets by searching the Gene Expression Omnibus (GEO) database, focusing on single-cell sequencing of normal mouse bladder within the past 5 years. Through quality control and batch effect elimination, we obtained a total of 24,930 cells including epithelial, stromal, and immune cells. Subgroup analysis, pseudotemporal analysis, cell–cell communication, and transcription factor analysis were conducted specifically on epithelial cells to identify a transitional state during basal cell differentiation. We further compared the expression profiles of key transcription factors in cancer and normal tissues. In addition, we also performed immunohistochemical staining and survival analysis for key transcription factors.ResultsSubgroup analysis revealed multiple subtypes of epithelial cells, including basal, umbrella, and intermediate cells. Through pseudotemporal analysis, we discovered the developmental trajectory from basal cells to umbrella cells and further found that Basal_I is a transitional state for basal cell differentiation. Cell-to-cell communication analyses highlighted the pivotal role of Basal_I in cell–cell interactions, and key ligand-receptor pairs associated with cancer progression were also identified. Furthermore, elevated expression levels of key transcription factors in Basal_I were found to be closely associated with the stage and prognosis of BC. Immunohistochemical staining results further confirmed the upregulated expression of these transcription factors in BC.ConclusionsCollectively, we found a transitional state of basal cells in normal bladder epithelial cells in mice, which may be related to the occurrence and development of BC, providing important clues for further understanding of the pathogenesis of BC. Our study provided possible molecular mechanisms or target for the research and treatment of BC.

Gene expression profiles of endothelium, microglia and oligodendrocytes in hippocampus of post-stroke depression rat at single cell resolution.

Post-stroke depression (PSD) is a common but severe mental complication after stroke. However, the cellular and molecular understanding of PSD is still yet to be illustrated. In current study, we prepared PSD rat model (MD) via unilateral middle cerebral artery occlusion (MCAO) and chronic stress stimulation (DEPR), and isolated hippocampal tissues for single cell sequencing of 10x Genomics Chromium. First, we determined the presence of the increased cell population of endothelium and microglia and the compromised oligodendrocytes in MD compared to NC, MCAO and DEPR. The enriched functions of highly variable genes (HVGs) of endothelium and microglia suggested a reinforced blood-brain barrier in MD. Next, cell clusters of endothelium, microglia and oligodendrocytes were individually analyzed, and the subtypes with distinct functions were identified. The presence of expression profiles, intercellular communications and signaling pathways of these three cell populations of PSD displayed a similar but more aggressive appearance with DEPR compared to MCAO and NC. Taken together, this study characterized the specific gene profile of endothelium, microglia and oligodendrocytes of hippocampal PSD by single cell sequencing, emphasizing the crosstalk among them to provide theoretical basis for the in-depth mechanism research and drug therapy of PSD.

Single-cell sequencing combined with transcriptomics and invivo and invitro analysis reveals the landscape offerroptosis in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a significant risk factor for acute myocardial infarction and is closely associated with ferroptosis. This study aimed to identify key ferroptosis-related genes as potential diagnostic markers for MIRI and to explore their roles in immune infiltration and therapeutic targeting in myocardial tissue. We obtained single-cell RNA sequencing (scRNA-seq) and RNA-seq data on MIRI from the GEO database, applied Seurat and UMAP for data processing and clustering, and analyzed ligand-receptor interactions using CellPhoneDB. By scoring ferroptosis in cardiomyocytes, we identified differentially expressed genes and conducted GO and KEGG pathway analyses. A protein interaction network was then constructed using the STRING database, and seven key genes (Atp5h, Vdac2, Pkm, Cycs, Hspa8, Tpi1, Ldha) were identified through Lasso regression modeling, showing significant associations with immune responses. Invivo experiments in a mouse ischemia-reperfusion model confirmed the roles of these seven genes in MIRI via RT-qPCR. To further investigate the role of Hspa8 in ferroptosis and MIRI, siRNA knockdown experiments were performed in H9C2 rat cardiomyocytes, and its involvement in ferroptosis was validated by JC-1 staining and PCR analysis. This study reveals the importance of ferroptosis-related genes in MIRI through integrated bioinformatics and experimental approaches, offering new insights into diagnostic markers and immune-related therapeutic targets for MIRI.

Combining Cre-LoxP and single-cell sequencing technologies: insights into the extracellular vesicle cargo transfer

The recent study from the Pogge von Strandmann group published in Cellular and Molecular Immunology , by Alashkar Alhamwe et al ., combined for the first time the Cre-LoxP recombination system with single-cell sequencing. The group monitored the tumor-derived extracellular vesicle (EV) uptake and the EV functions in the recipient non-malignant cells in a pancreatic ductal adenocarcinoma mouse model. Recombination events and EV uptake, together with resulting gene expression changes in macrophages, neutrophils, and mast cells, were detected by single-cell sequencing technology of the tumor tissue. This new approach is highly specific, as it can identify single EV recipient cells without interfering with the EV biogenesis or the phenotype.

Wnt 3a-Modified Scaffolds Improve Nerve Regeneration by Boosting Schwann Cell Function.

A pivotal approach in engineering artificial peripheral nerve sheaths encompasses the augmentation of the regenerative microenvironment via the manipulation of Schwann cells (SCs). Our investigation employed single-cell sequencing analysis to elucidate the potential functions of Schwann cells and the Wnt pathway in facilitating peripheral nerve regeneration. In vitro studies showed that activating the Wnt signaling pathway promotes the transition to repair SCs, boosting their growth, movement, and immune functions. To better understand the peripheral nerve regeneration environment, we created a polymer scaffold using ammonization and electrospinning. The Wnt3a protein was incorporated into the polycaprolactone (PCL) electrospun fiber surface. In a rat sciatic nerve defect model, the Wnt3a-modified scaffold showed better nerve repair outcomes than traditional electrospun scaffolds. After a week, the test group showed better immune regulation and angiogenesis, with a significant increase in axon growth rate observed after 3 weeks. Three-month-long animal experiments revealed notable improvements in neuroelectrophysiology, reduced organ atrophy, and enhanced sciatic nerve recovery. In this nerve defect model, Wnt3a-modified neural scaffolds achieved repair effects.

Multi-omics analysis of Prolyl 3-hydroxylase 1 as a prognostic biomarker for immune infiltration in ccRCC

The formation of human collagen requires the presence of Prolyl 3-hydroxylase 1 (P3H1), but the regulatory mechanism of P3H1 remained insufficiently understood. Our study aimed to identify the role of P3H1 in clear cell renal cell carcinoma (ccRCC). P3H1 expression in ccRCC was validated using multiple databases and in vitro experiments. We performed a correlation analysis of P3H1 with drug sensitivity, immune checkpoints, and immune cell infiltration using transcriptome and single-cell sequencing. Drawing upon the Encyclopedia of RNA Interactomes database, we selected P3H1 as the focal point of our investigation, meticulously uncovering the intricate network of microRNAs and lncRNAs that potentially orchestrate ceRNA mechanisms. This study employs a multidimensional approach integrating vitro assays and multi-omics bioinformatics analyses to investigate P3H1’s impact on ccRCC prognosis, immune modulation, immune checkpoints, ceRNA regulatory network, drug sensitivity, and therapeutic responses, aiming to uncover new insights into its therapeutic potential and inform future clinical strategies.

Single-cell sequencing reveals cellular heterogeneity of nucleus pulposus in intervertebral disc degeneration

The nucleus pulposus (NP) plays a vital role in intervertebral disc degeneration (IVDD). Previous studies have revealed cellular heterogeneity in NP tissue during IVDD progression. However, the cellular and molecular alterations of diverse cell clusters during IVDD remain to be fully elucidated. NP tissues were isolated from patients with different grades of IVDD undergoing discectomy, and then subjected to single-cell RNA sequencing (scRNA-seq). Cell subsets were identified based on unbiased clustering of gene expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine the molecular features of diverse cell clusters. Monocle analysis was used to illustrate the differentiation trajectories of chondrocytes. Additionally, CellPhoneDB analysis revealed potential interactions between chondrocytes and other cells during IVDD. Based on the expression profiles of 47,610 individual cells, eight putative clusters including chondrocytes, endothelial cells, fibroblasts, macrophages, mural cells, osteoclasts, proliferating stromal cells and T cells were identified. The chondrocyte cluster was classified into three subsets, C1-C3, which were associated with stress-resistance, fibrosis and inflammatory responses, respectively. Pseudo-time trajectories suggested that chondrocytes gradually differentiated into fibroblasts during IVDD. Immune cells including cDC2s, macrophages and monocytes were identified. Further analysis showed that chondrocytes might communicate with immune cells via the MIF, TNFSF9, SPP1 and CCL4L2 signaling pathways. In addition, we found that invading endothelial cells might interact with chondrocytes through the COL4A1, CXCL12, VEGFA and SEMA3E signaling pathways. Our results reveal the cellular complexity and phenotypic characteristics of NP tissues at single-cell resolution, which will contribute to the in-depth investigation of preventative and regenerative strategies for IVDD.

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

Reproductomics: Exploring the Applications and Advancements of Computational Tools

Over recent decades, advancements in omics technologies, such as proteomics, genomics, epigenomics, metabolomics, transcriptomics, and microbiomics, have significantly enhanced our understanding of the molecular mechanisms underlying various physiological and pathological processes. Nonetheless, the analysis and interpretation of vast omics data concerning reproductive diseases are complicated by the cyclic regulation of hormones and multiple other factors, which, in conjunction with a genetic makeup of an individual, lead to diverse biological responses. Reproductomics investigates the interplay between a hormonal regulation of an individual, environmental factors, genetic predisposition (DNA composition and epigenome), health effects, and resulting biological outcomes. It is a rapidly emerging field that utilizes computational tools to analyze and interpret reproductive data, with the aim of improving reproductive health outcomes. It is time to explore the applications of reproductomics in understanding the molecular mechanisms underlying infertility, identification of potential biomarkers for diagnosis and treatment, and in improving assisted reproductive technologies (ARTs). Reproductomics tools include machine learning algorithms for predicting fertility outcomes, gene editing technologies for correcting genetic abnormalities, and single cell sequencing techniques for analyzing gene expression patterns at the individual cell level. However, there are several challenges, limitations and ethical issues involved with the use of reproductomics, such as the applications of gene editing technologies and their potential impact on future generations are discussed. The review comprehensively covers the applications and advancements of reproductomics, highlighting its potential to improve reproductive health outcomes and deepen our understanding of reproductive molecular mechanisms.

Identifying optimal tumor-associated antigen combinations with single-cell genomics to enable multi-targeting therapies

Identifying optimal tumor-associated antigen combinations with single-cell genomics to enable multi-targeting therapies

Inorganic pyrophosphatase 1: a key player in immune and metabolic reprogramming in ankylosing spondylitis.

The relationships among immune cells, metabolites, and AS events were analyzed via Mendelian randomization (MR), and potential immune cells and metabolites were identified as risk factors for AS. Their relationships were subjected to intermediary MR analysis to identify the final immune cells and metabolites. The vertebral bone marrow blood samples from three patients with and without AS were subjected to 10× single-cell sequencing to further elucidate the role of immune cells in AS. The key genes were screened via expression quantitative trait loci (eQTLs) and MR analyses. The metabolic differences between the two groups were compared through single-cell metabolism analysis. Two subgroups of differentiated (CD)8+ memory T cells and naive B cells were obtained from the combined results of intermediary MR analysis and AS single-cell analysis. After the verification of key genes, inorganic pyrophosphatase 1 (PPA1) was identified as the hub gene, as it is differentially expressed in CD8+ memory T cells and can affect the metabolism of T cells in AS by affecting the expression of ferulic acid (FA)4 sulfate, which participates in the cellular immunity in AS.

Integration of single-cell and spatial transcriptome sequencing identifies CDKN2A as a senescent biomarker in endothelial cells implicating hepatocellular carcinoma malignancy.

Highly complex tumor microenvironment makes hepatocellular carcinoma (HCC) as one of the most malignant tumors worldwide. The role of cellular senescence in HCC has been gradually recognized. The present study aimed to comprehensively elucidate the senescence-related features of HCC in single-cell and spatial dimension. Single-cell RNA sequencing (scRNA-Seq) data was used to clarify the heterogeneity of senescence-related genes (SRGs) among multiple cell types within HCC. Spatial transcriptome RNA sequencing (stRNA-Seq) data was used for depicting SRGs features in spatial dimension. A prognostic model based on SRGs was constructed by using of bulk sequencing (bulk-Seq) data of HCC. The cell-cell interaction of senescent endothelial cells (ECs) in tumor microenvironment was analyzed. Then, the role of senescent ECs was verified through in vitro and in vivo experiments. The level of senescence demonstrated substantial heterogeneity among different cell types within tumor microenvironment of HCC, where ECs exhibited the most prominent senescent phenotype. Senescent ECs activated specific regulatory pathways through communicating with other cell types, with a potential impact on tumor progression. Spatial analysis revealed senescent ECs mainly located in the core region of HCC. The interaction of senescent ECs and immune cells implicated their role in tumor progression and immunotherapeutic response. In addition, CDKN2A was identified as an independent risk factor for HCC prognosis by constructing a prognostic model. Patients with high risk displayed an even worse outcome. The experimental verification indicated senescence of ECs determined by CDKN2A exhibited a secretory phenotype. Furthermore, senescent ECs with CDKN2A overexpression promote the proliferation and migration of HCC. The present study recognizes the critical effect of senescent ECs defined by CDKN2A in the promotion of tumor progression, which sheds new light on the investigation of ECs senescence in HCC.