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Transcriptome-wide analysis of differential expression in perturbation atlases.
Single-cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are noisy, and many effects may go undetected. Here we introduce transcriptome-wide analysis of differential expression (TRADE)-a statistical model for the distribution of true differential expression effects that accounts for estimation error appropriately. TRADE estimates the 'transcriptome-wide impact', which quantifies the total effect of a perturbation across the transcriptome. Analyzing several large Perturb-seq datasets, we show that many transcriptional effects remain undetected in standard analyses but emerge in aggregate using TRADE. A typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene affects over 500. We find moderate consistency of perturbation effects across cell types, identify perturbations where transcriptional responses vary qualitatively across dosage levels and clarify the relationship between genetic and transcriptomic correlations across neuropsychiatric disorders.
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Single-nuclei transcriptome analysis of IgM+ cells isolated from channel catfish (Ictalurus punctatus) spleen.
Catfish production is the primary aquaculture sector in the United States, and the key cultured species is channel catfish (Ictalurus punctatus). The major causes of production losses are pathogenic diseases, and the spleen, an important site of adaptive immunity, is implicated in these diseases. To examine the channel catfish immune system, single-nuclei transcriptomes of sorted and captured IgM+ cells were produced from adult channel catfish. Three channel catfish (~1 kg) were euthanized, the spleen dissected, and the tissue dissociated. The lymphocytes were isolated using a Ficoll gradient and IgM+ cells were then sorted with flow cytometry. The IgM+ cells were lysed and single-nuclei libraries generated using a Chromium Next GEM Single Cell 3' GEM Kit and the Chromium X Instrument (10x Genomics) and sequenced with the Illumina NovaSeq X Plus sequencer. The reads were aligned to the I. punctatus reference assembly (Coco_2.0) using Cell Ranger, and normalization, cluster analysis, and differential gene expression analysis were carried out with Seurat. Across the three samples, approximately 753.5 million reads were generated for 18,686 cells. After filtering, 10,637 cells remained for the cluster analysis. The cluster analysis identified 16 clusters which were classified as B cells (10,276), natural killer-like (NK-like) cells (178), T cells or natural killer cells (45), hematopoietic stem and progenitor cells (HSPC)/megakaryocytes (MK) (66), myeloid/epithelial cells (40), and plasma cells (32). The B cell clusters were further defined as different populations of mature B cells, cycling B cells, and plasma cells. The plasma cells highly expressed ighm and we demonstrated that the secreted form of the transcript was largely being expressed by these cells. This atlas provides insight into the gene expression of IgM+ immune cells in channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells.
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Single-cell transcriptomic profiling of colorectal cancer brain metastasis.
BACKGROUND: Brain metastasis from colorectal cancer (CRC) remains a highly lethal disease. However, the molecular mechanisms driving CRC brain metastasis are not fully characterized. METHODS: Employing single-cell RNA sequencing (scRNA-seq), we delineated the cellular atlas of a resected CRC brain metastasis specimen and particularly focused on the tumor cell compartment. RESULTS: Our scRNA-seq analysis profiled 4,748 high-quality single cells from a CRC brain metastasis sample. Tumor cells constituted the dominant population. Within the immune compartment, myeloid cells, including macrophages and monocytes, represented the predominant components of the metastatic microenvironment. In addition, key elements of the blood-tumor barrier, such as endothelial cells and pericytes, were characterized and implicated in brain metastasis. Tumor cells showed phenotypic plasticity, with transcriptional dynamics consistent with a mesenchymal-to-epithelial transition. Notably, a highly proliferative tumor cell subset with elevated KMT2B expression was identified. Functional analyses demonstrated that KMT2B depletion significantly impaired CRC cell proliferation and migration. Mechanistically, KMT2B promoted metastatic potential partly by upregulating MEX3A expression. Furthermore, high expression levels of KMT2B and MEX3A were associated with poor prognosis in both CRC and pan-cancer patients. CONCLUSION: Collectively, this study identifies KMT2B as a potential driver of CRC brain metastasis and highlights it as a candidate therapeutic target for CRC treatment.
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A single-cell atlas of the myometrium in human parturition.
Parturition is a well-orchestrated process characterized by increased uterine contractility, cervical ripening, and activation of the chorioamniotic membranes; yet, the transition from a quiescent to a contractile myometrium heralds the onset of labor. However, the cellular underpinnings of human parturition in the uterine tissues are still poorly understood. Herein, we performed a comprehensive study of the human myometrium during spontaneous term labor using single-cell RNA sequencing (scRNA-Seq). First, we established a single-cell atlas of the human myometrium and unraveled the cell type-specific transcriptomic activity modulated during labor. Major cell types included distinct subsets of smooth muscle cells, monocytes/macrophages, stromal cells, and endothelial cells, all of which communicated and participated in immune (e.g., inflammation) and nonimmune (e.g., contraction) processes associated with labor. Furthermore, integrating scRNA-Seq and microarray data with deconvolution of bulk gene expression highlighted the contribution of smooth muscle cells to labor-associated contractility and inflammatory processes. Last, myometrium-derived single-cell signatures can be quantified in the maternal whole-blood transcriptome throughout pregnancy and are enriched in women in labor, providing a potential means of noninvasively monitoring pregnancy and its complications. Together, our findings provide insights into the contributions of specific myometrial cell types to the biological processes that take place during term parturition.
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Single-Cell Transcriptome Atlas and Dynamic Regulatory Mechanisms of Anther Development in Alfalfa (Medicago sativa L.).
Anthers consist of various specialised cell types and play a significant role in plant reproduction. Although the molecular mechanisms underlying anther development and regulation have been extensively studied, the single-cell transcriptional landscape and dynamic regulation during anther development in M. sativa remain largely unexplored. In the present study, we constructed the first single-cell transcriptome atlas of M. sativa anthers to provide a comprehensive view of cell type-specific gene expression and epigenetic modifications. The reconstruction of the developmental trajectories of tapetum and microspores led to the identification of novel genes and elucidated the regulatory networks involved in tapetum formation. Our findings revealed rapid functional transitions in tapetum during the tetrad stage, including cell formation, specialisation, and programmed cell death (PCD). Additionally, we analysed the stages at which tapetal degradation and microspore shrinkage occurred in the sterile line. Overall, this study offers valuable insights into the molecular mechanisms underlying anther development in M. sativa. Specifically, MsKIN14P was identified as a key regulator of microtubules and the cytoskeleton during mitosis, and the transcription factors ERF3 and ERF025 were shown to influence anther development through the ethylene response pathways. These findings provide an essential theoretical foundation for the development of novel male-sterile lines and enhance the breeding capabilities of M. sativa.
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Single-cell RNA sequencing reveals cell type- and artery type-specific vascular remodelling in male spontaneously hypertensive rats.
AIMS: Hypertension is a major risk factor for cardiovascular diseases. However, vascular remodelling, a hallmark of hypertension, has not been systematically characterized yet. We described systematic vascular remodelling, especially the artery type- and cell type-specific changes, in hypertension using spontaneously hypertensive rats (SHRs). METHODS AND RESULTS: Single-cell RNA sequencing was used to depict the cell atlas of mesenteric artery (MA) and aortic artery (AA) from SHRs. More than 20 000 cells were included in the analysis. The number of immune cells more than doubled in aortic aorta in SHRs compared to Wistar Kyoto controls, whereas an expansion of MA mesenchymal stromal cells (MSCs) was observed in SHRs. Comparison of corresponding artery types and cell types identified in integrated datasets unravels dysregulated genes specific for artery types and cell types. Intersection of dysregulated genes with curated gene sets including cytokines, growth factors, extracellular matrix (ECM), receptors, etc. revealed vascular remodelling events involving cell-cell interaction and ECM re-organization. Particularly, AA remodelling encompasses upregulated cytokine genes in smooth muscle cells, endothelial cells, and especially MSCs, whereas in MA, change of genes involving the contractile machinery and downregulation of ECM-related genes were more prominent. Macrophages and T cells within the aorta demonstrated significant dysregulation of cellular interaction with vascular cells. CONCLUSION: Our findings provide the first cell landscape of resistant and conductive arteries in hypertensive animal models. Moreover, it also offers a systematic characterization of the dysregulated gene profiles with unbiased, artery type-specific and cell type-specific manners during hypertensive vascular remodelling.
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Single-cell transcriptional atlas of tumor-associated macrophages in breast cancer.
BACKGROUND: The internal heterogeneity of breast cancer, notably the tumor microenvironment (TME) consisting of malignant and non-malignant cells, has been extensively explored in recent years. The cells in this complex cellular ecosystem activate or suppress tumor immunity through phenotypic changes, secretion of metabolites and cell-cell communication networks. Macrophages, as the most abundant immune cells within the TME, are recruited by malignant cells and undergo phenotypic remodeling. Tumor-associated macrophages (TAMs) exhibit a variety of subtypes and functions, playing significant roles in impacting tumor immunity. However, their precise subtype delineation and specific function remain inadequately defined. METHODS: The publicly available single-cell transcriptomes of 49,141 cells from eight breast cancer patients with different molecular subtypes and stages were incorporated into our study. Unsupervised clustering and manual cell annotation were employed to accurately classify TAM subtypes. We then conducted functional analysis and constructed a developmental trajectory for TAM subtypes. Subsequently, the roles of TAM subtypes in cell-cell communication networks within the TME were explored using endothelial cells (ECs) and T cells as key nodes. Finally, analyses were repeated in another independent publish scRNA datasets to validate our findings for TAM characterization. RESULTS: TAMs are accurately classified into 7 subtypes, displaying anti-tumor or pro-tumor roles. For the first time, we identified a new TAM subtype capable of proliferation and expansion in breast cancer-TUBA1B+ TAMs playing a crucial role in TAMs diversity and tumor progression. The developmental trajectory illustrates how TAMs are remodeled within the TME and undergo phenotypic and functional changes, with TUBA1B+ TAMs at the initial point. Notably, the predominant TAM subtypes varied across different molecular subtypes and stages of breast cancer. Additionally, our research on cell-cell communication networks shows that TAMs exert effects by directly modulating intrinsic immunity, indirectly regulating adaptive immunity through T cells, as well as influencing tumor angiogenesis and lymphangiogenesis through ECs. CONCLUSIONS: Our study establishes a precise single-cell atlas of breast cancer TAMs, shedding light on their multifaceted roles in tumor biology and providing resources for targeting TAMs in breast cancer immunotherapy.
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A single-cell analysis of breast cancer cell lines to study tumour heterogeneity and drug response.
Cancer cells within a tumour have heterogeneous phenotypes and exhibit dynamic plasticity. How to evaluate such heterogeneity and its impact on outcome and drug response is still unclear. Here, we transcriptionally profile 35,276 individual cells from 32 breast cancer cell lines to yield a single cell atlas. We find high degree of heterogeneity in the expression of biomarkers. We then train a deconvolution algorithm on the atlas to determine cell line composition from bulk gene expression profiles of tumour biopsies, thus enabling cell line-based patient stratification. Finally, we link results from large-scale in vitro drug screening in cell lines to the single cell data to computationally predict drug responses starting from single-cell profiles. We find that transcriptional heterogeneity enables cells with differential drug sensitivity to co-exist in the same population. Our work provides a framework to determine tumour heterogeneity in terms of cell line composition and drug response.
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Spatial and single-cell transcriptome analysis reveals changes in gene expression in response to drug perturbation in rat kidney.
The kidney is a complex organ that consists of various types of cells. It is occasionally difficult to resolve molecular alterations and possible perturbations that the kidney experiences due to drug-induced damage. In this study, we performed spatial and single-cell transcriptome analysis of rat kidneys and constructed a precise rat renal cell atlas with spatial information. Using the constructed catalogue, we were able to characterize cells of several minor populations, such as macula densa or juxtaglomerular cells. Further inspection of the spatial gene expression data allowed us to identify the upregulation of genes involved in the renin regulating pathway in losartan-treated populations. Losartan is an angiotensin II receptor antagonist drug, and the observed upregulation of the renin pathway-related genes could be due to feedback from the hypotensive action of the drug. Furthermore, we found spatial heterogeneity in the response to losartan among the glomeruli. These results collectively indicate that integrated single-cell and spatial gene expression analysis is a powerful approach to reveal the detailed associations between the different cell types spanning the complicated renal compartments.
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Developing an epithelial signature for prognosis of papillary thyroid carcinoma.
Papillary thyroid carcinoma (PTC) shows heterogeneous clinical behavior, and current clinicopathologic factors do not fully capture epithelial aggressiveness or tumor microenvironment (TME) remodeling. We integrated single-cell RNA sequencing (scRNA-seq) datasets from normal thyroid tissue, primary PTC, and lymph-node metastases to construct a cross-stage cellular atlas and to characterize epithelial and T/NK cell heterogeneity. Guided by the malignant epithelial program identified at single-cell resolution, we developed a four-gene risk signature in the TCGA-THCA cohort using Cox and LASSO regression. We further evaluated pathway activity, immune and stromal features, T cell receptor (TCR) diversity, and exploratory in silico drug-response patterns, and we performed small interfering RNA (siRNA)-mediated knockdown of TMEM45A and STC1 in PTC cell lines. The integrated atlas revealed stage-related remodeling of epithelial, immune, and stromal compartments and identified a malignant epithelial state enriched for proliferation-, stress-, and migration-related programs. The four-gene signature was associated with overall survival stratification in TCGA-THCA after adjustment for age, sex, and stage. The high-risk group showed reduced immune infiltration, stronger stromal and epithelial-mesenchymal transition (EMT) signals, lower TCR diversity, and distinct predicted drug-response patterns. In vitro, TMEM45A or STC1 silencing suppressed proliferation and migration and altered EMT-related markers in PTC cells. These findings indicate that a malignant epithelial program defined by single-cell analysis can be translated into a four-gene signature associated with epithelial aggressiveness and TME remodeling in PTC, although its clinical utility requires validation in independent cohorts.