scholarly journals Single-cell RNA sequencing reveals heterogeneous tumor and immune cell populations in early-stage lung adenocarcinomas harboring EGFR mutations

Oncogene ◽  
2020 ◽  
Author(s):  
Di He ◽  
Di Wang ◽  
Ping Lu ◽  
Nan Yang ◽  
Zhigang Xue ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Early Stage ◽  
Cell Types ◽  
Egfr Mutations ◽  
Advanced Tumor ◽  
Single Cell Rna Sequencing

Abstract Lung adenocarcinoma (LUAD) harboring EGFR mutations prevails in Asian population. However, the inter-patient and intra-tumor heterogeneity has not been addressed at single-cell resolution. Here we performed single-cell RNA sequencing (scRNA-seq) of total 125,674 cells from seven stage-I/II LUAD samples harboring EGFR mutations and five tumor-adjacent lung tissues. We identified diverse cell types within the tumor microenvironment (TME) in which myeloid cells and T cells were the most abundant stromal cell types in tumors and adjacent lung tissues. Within tumors, accompanied by an increase in CD1C+ dendritic cells, the tumor-associated macrophages (TAMs) showed pro-tumoral functions without signature gene expression of defined M1 or M2 polarization. Tumor-infiltrating T cells mainly displayed exhausted and regulatory T-cell features. The adenocarcinoma cells can be categorized into different subtypes based on their gene expression signatures in distinct pathways such as hypoxia, glycolysis, cell metabolism, translation initiation, cell cycle, and antigen presentation. By performing pseudotime trajectory, we found that ELF3 was among the most upregulated genes in more advanced tumor cells. In response to secretion of inflammatory cytokines (e.g., IL1B) from immune infiltrates, ELF3 in tumor cells was upregulated to trigger the activation of PI3K/Akt/NF-κB pathway and elevated expression of proliferation and anti-apoptosis genes such as BCL2L1 and CCND1. Taken together, our study revealed substantial heterogeneity within early-stage LUAD harboring EGFR mutations, implicating complex interactions among tumor cells, stromal cells and immune infiltrates in the TME.

scholarly journals Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

Cell Reports ◽  
2014 ◽  
Vol 8 (6) ◽  
pp. 1905-1918 ◽  
Author(s):  
David T. Ting ◽  
Ben S. Wittner ◽  
Matteo Ligorio ◽  
Nicole Vincent Jordan ◽  
Ajay M. Shah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Circulating Tumor Cells ◽  
Matrix Gene ◽  
Single Cell Rna Sequencing

scholarly journals Power Analysis of Single Cell RNA-Sequencing Experiments

2016 ◽  
Author(s):  
Valentine Svensson ◽  
Kedar Nath Natarajan ◽  
Lam-Ha Ly ◽  
Ricardo J Miragaia ◽  
Charlotte Labalette ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Developmental Process ◽  
Cell Types ◽  
Rna Degradation ◽  
Published Data ◽  
Minute Amount ◽  
Trade Offs ◽  
Single Cell Rna Sequencing

AbstractHigh-throughput single cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, and has revealed new cell types, and new insights into developmental process and stochasticity in gene expression. There are now several published scRNA-seq protocols, which all sequence transcriptomes from a minute amount of starting material. Therefore, a key question is how these methods compare in terms of sensitivity of detection of mRNA molecules, and accuracy of quantification of gene expression. Here, we assessed the sensitivity and accuracy of many published data sets based on standardized spike-ins with a uniform raw data processing pipeline. We developed a flexible and fast UMI counting tool (https://github.com/vals/umis) which is compatible with all UMI based protocols. This allowed us to relate these parameters to sequencing depth, and discuss the trade offs between the different methods. To confirm our results, we performed experiments on cells from the same population using three different protocols. We also investigated the effect of RNA degradation on spike-in molecules, and the average efficiency of scRNA-seq on spike-in molecules versus endogenous RNAs.

scholarly journals Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiuying Li ◽  
Guillaume Noell ◽  
Tracy Tabib ◽  
Alyssa D. Gregory ◽  
Humberto E. Trejo Bittar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Single Cell Rna Sequencing ◽  
Ciliated Epithelial Cells ◽  
Severe Copd

Abstract Background Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. Methods To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. Results T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

scholarly journals Identification of silkworm hemocyte subsets and analysis of their response to BmNPV infection based on single-cell RNA sequencing

2020 ◽  
Author(s):  
Min Feng ◽  
Junming Xia ◽  
Shigang Fei ◽  
Xiong Wang ◽  
Yaohong Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Current Knowledge ◽  
Early Stage ◽  
Expression Profiles ◽  
Marker Genes ◽  
Potential Marker ◽  
Wide Range ◽  
Single Cell Rna Sequencing

AbstractA wide range of hemocyte types exist in insects but a full definition of the different subclasses is not yet established. The current knowledge of the classification of silkworm hemocytes mainly comes from morphology rather than specific markers, so our understanding of the detailed classification, hemocyte lineage and functions of silkworm hemocytes is very incomplete. Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of the baculoviruses, which are a major pathogens that specifically infects silkworms and cause serious loss in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of silkworm hemocytes in BmNPV and mock-infected larvae to comprehensively identify silkworm hemocyte subsets and determined specific molecular and cellular characteristics in each hemocyte subset before and after viral infection. A total of 19 cell clusters and their potential marker genes were identified in silkworm hemocytes. Among these hemocyte clusters, clusters 0, 1, 2, 5 and 9 might be granulocytes (GR); clusters 14 and 17 were predicted as plasmatocytes (PL); cluster 18 was tentatively identified as spherulocytes (SP); and clusters 7 and 11 could possibly correspond to oenocytoids (OE). In addition, all of the hemocyte clusters were infected by BmNPV and some infected cells carried high viral-load in silkworm larvae at 3 day post infection (dpi). Interestingly, BmNPV infection can cause severe and diverse changes in gene expression in hemocytes. Cells belonging to the infection group mainly located at the early stage of the pseudotime trajectories. Furthermore, we found that BmNPV infection suppresses the immune response in the major hemocyte types. In summary, our scRNA-seq analysis revealed the diversity of silkworm hemocytes and provided a rich resource of gene expression profiles for a systems-level understanding of their functions in the uninfected condition and as a response to BmNPV.

scholarly journals Single-Cell RNA Sequencing Reveals Multiple Pathways and the Tumor Microenvironment Could Lead to Chemotherapy Resistance in Cervical Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Meijia Gu ◽  
Ti He ◽  
Yuncong Yuan ◽  
Suling Duan ◽  
Xin Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cervical Cancer ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cancer Biology ◽  
Chemotherapy Resistance ◽  
Cell Types ◽  
Functional Enrichment ◽  
Single Cell Rna Sequencing

BackgroundCervical cancer is one of the most common gynecological cancers worldwide. The tumor microenvironment significantly influences the therapeutic response and clinical outcome. However, the complex tumor microenvironment of cervical cancer and the molecular mechanisms underlying chemotherapy resistance are not well studied. This study aimed to comprehensively analyze cells from pretreated and chemoresistant cervical cancer tissues to generate a molecular census of cell populations.MethodsBiopsy tissues collected from patients with cervical squamous cell carcinoma, cervical adenocarcinoma, and chronic cervicitis were subjected to single-cell RNA sequencing using the 10× Genomics platform. Unsupervised clustering analysis of cells was performed to identify the main cell types, and important cell clusters were reclustered into subpopulations. Gene expression profiles and functional enrichment analysis were used to explore gene expression and functional differences between cell subpopulations in cervicitis and cervical cancer samples and between chemoresistant and chemosensitive samples.ResultsA total of 24,371 cells were clustered into nine separate cell types, including immune and non-immune cells. Differentially expressed genes between chemoresistant and chemosensitive patients enriched in the phosphoinositide 3-kinase (PI3K)/AKT pathway were involved in tumor development, progression, and apoptosis, which might lead to chemotherapy resistance.ConclusionsOur study provides a comprehensive overview of the cancer microenvironment landscape and characterizes its gene expression and functional difference in chemotherapy resistance. Consequently, our study deepens the insights into cervical cancer biology through the identification of gene markers for diagnosis, prognosis, and therapy.

scholarly journals Impact of sequencing depth and read length on single cell RNA sequencing data: lessons from T cells

2017 ◽  
Author(s):  
Simone Rizzetto ◽  
Auda A. Eltahla ◽  
Peijie Lin ◽  
Rowena Bull ◽  
Andrew R. Lloyd ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Sequencing Depth ◽  
Read Length ◽  
Single Cell Rna Sequencing

ABSTRACTSingle cell RNA sequencing (scRNA-seq) has shown great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant sub-populations of T cells, and notably the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, such as RNA library capture, cell quality, and sequencing output have been suggested to affect the quality of scRNA-seq data, but these factors have not been systematically examined.We studied the effect of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. TCRαβ were detected in 1,027 cells (79%), with a success rate between 81% and 100% for datasets with at least 250,000 (PE) reads of length >50 bp.Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

scholarly journals Glyoxal as alternative fixative for single cell RNA sequencing

2021 ◽  
Author(s):  
Josephine Bageritz ◽  
Niklas Krausse ◽  
Schayan Yousefian ◽  
Svenja Leible ◽  
Erica Valentini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cell Isolation ◽  
Sample Collection ◽  
Experimental Conditions ◽  
Single Cell Rna Sequencing ◽  
Highly Expressed Genes ◽  
Single Cell Isolation

Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.

STEM-27. A PERIVASCULAR STEM CELL UNDERLIES VERTEBRATE MENINGEAL TUMORIGENESIS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi26-vi27
Author(s):  
Abrar Choudhury ◽  
Martha Cady ◽  
Calixto Lucas ◽  
Brisa Palikuqi ◽  
Ophir Klein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Single Cells ◽  
Cell Types ◽  
Human Meningioma ◽  
Single Cell Rna Sequencing ◽  
Human Meningiomas

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

scholarly journals Single‐cell RNA sequencing in cancer research

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Yijie Zhang ◽  
Dan Wang ◽  
Miao Peng ◽  
Le Tang ◽  
Jiawei Ouyang ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Immune Cells ◽  
Immune Escape ◽  
Tumor Development ◽  
Cell Types ◽  
Resistance Mechanisms ◽  
Single Cell Rna Sequencing ◽  
Tumor Research

AbstractSingle-cell RNA sequencing (scRNA-seq), a technology that analyzes transcriptomes of complex tissues at single-cell levels, can identify differential gene expression and epigenetic factors caused by mutations in unicellular genomes, as well as new cell-specific markers and cell types. scRNA-seq plays an important role in various aspects of tumor research. It reveals the heterogeneity of tumor cells and monitors the progress of tumor development, thereby preventing further cellular deterioration. Furthermore, the transcriptome analysis of immune cells in tumor tissue can be used to classify immune cells, their immune escape mechanisms and drug resistance mechanisms, and to develop effective clinical targeted therapies combined with immunotherapy. Moreover, this method enables the study of intercellular communication and the interaction of tumor cells and non-malignant cells to reveal their role in carcinogenesis. scRNA-seq provides new technical means for further development of tumor research and is expected to make significant breakthroughs in this field. This review focuses on the principles of scRNA-seq, with an emphasis on the application of scRNA-seq in tumor heterogeneity, pathogenesis, and treatment.

scholarly journals Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Katharina Rindler ◽  
Wolfgang M. Bauer ◽  
Constanze Jonak ◽  
Matthias Wielscher ◽  
Lisa E. Shaw ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Mycosis Fungoides ◽  
Cell Receptor ◽  
Skin Tumor ◽  
Single Cell Rna Sequencing

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

Sign in / Sign up

Export Citation Format

Share Document