Single-Cell RNA Sequencing of a Postmenopausal Normal Breast Tissue Identifies Multiple Cell Types That Contribute to Breast Cancer

The human breast is composed of diverse cell types. Studies have delineated mammary epithelial cells, but the other cell types in the breast have scarcely been characterized. In order to gain insight into the cellular composition of the tissue, we performed droplet-mediated RNA sequencing of 3193 single cells isolated from a postmenopausal breast tissue without enriching for epithelial cells. Unbiased clustering analysis identified 10 distinct cell clusters, seven of which were nonepithelial devoid of cytokeratin expression. The remaining three cell clusters expressed cytokeratins (CKs), representing breast epithelial cells; Cluster 2 and Cluster 7 cells expressed luminal and basal CKs, respectively, whereas Cluster 9 cells expressed both luminal and basal CKs, as well as other CKs of unknown specificity. To assess which cell type(s) potentially contributes to breast cancer, we used the differential gene expression signature of each cell cluster to derive gene set variation analysis (GSVA) scores and classified breast tumors in The Cancer Gene Atlas (TGGA) dataset (n = 1100) by assigning the highest GSVA scoring cell cluster number for each tumor. The results showed that five clusters (Clusters 2, 3, 7, 8, and 9) could categorize >85% of breast tumors collectively. Notably, Cluster 2 (luminal epithelial) and Cluster 3 (fibroblast) tumors were equally prevalent in the luminal breast cancer subtypes, whereas Cluster 7 (basal epithelial) and Cluster 9 (other epithelial) tumors were present primarily in the triple-negative breast cancer (TNBC) subtype. Cluster 8 (immune) tumors were present in all subtypes, indicating that immune cells may contribute to breast cancer regardless of the subtypes. Cluster 9 tumors were significantly associated with poor patient survival in TNBC, suggesting that this epithelial cell type may give rise to an aggressive TNBC subset.

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

Respiratory Research ◽

10.1186/s12931-021-01675-2 ◽

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.

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Single-Cell Analysis of the Gene Expression Effects of Developmental Lead (Pb) Exposure on the Mouse Hippocampus

Toxicological Sciences ◽

10.1093/toxsci/kfaa069 ◽

2020 ◽

Vol 176 (2) ◽

pp. 396-409

Author(s):

Kelly M Bakulski ◽

John F Dou ◽

Robert C Thompson ◽

Christopher Lee ◽

Lauren Y Middleton ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Cell Analysis ◽

Expression Patterns ◽

Cell Types ◽

Cell Cluster ◽

Marker Genes ◽

Cell Type ◽

Cell Clusters ◽

Pb Exposure

Abstract Lead (Pb) exposure is ubiquitous with permanent neurodevelopmental effects. The hippocampus brain region is involved in learning and memory with heterogeneous cellular composition. The hippocampus cell type-specific responses to Pb are unknown. The objective of this study is to examine perinatal Pb treatment effects on adult hippocampus gene expression, at the level of individual cells. In mice perinatally exposed to control water or a human physiologically relevant level (32 ppm in maternal drinking water) of Pb, 2 weeks prior to mating through weaning, we tested for hippocampus gene expression and cellular differences at 5 months of age. We sequenced RNA from 5258 hippocampal cells to (1) test for treatment gene expression differences averaged across all cells, (2) compare cell cluster composition by treatment, and (3) test for treatment gene expression and pathway differences within cell clusters. Gene expression patterns revealed 12 hippocampus cell clusters, mapping to major expected cell types (eg, microglia, astrocytes, neurons, and oligodendrocytes). Perinatal Pb treatment was associated with 12.4% more oligodendrocytes (p = 4.4 × 10−21) in adult mice. Across all cells, Pb treatment was associated with expression of cell cluster marker genes. Within cell clusters, Pb treatment (q < 0.05) caused differential gene expression in endothelial, microglial, pericyte, and astrocyte cells. Pb treatment upregulated protein folding pathways in microglia (p = 3.4 × 10−9) and stress response in oligodendrocytes (p = 3.2 × 10−5). Bulk tissue analysis may be influenced by changes in cell type composition, obscuring effects within vulnerable cell types. This study serves as a biological reference for future single-cell toxicant studies, to ultimately characterize molecular effects on cognition and behavior.

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Single Cell RNA Sequencing Identifies IGFBP5 And QKI In Ciliated Epithelial Cell Genes Associated With Severe COPD

10.21203/rs.3.rs-100834/v1 ◽

2020 ◽

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 to identify transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

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Normal Cell-Type Epigenetics and Breast Cancer Classification: A Case Study of Cell Mixture–Adjusted Analysis of DNA Methylation Data from Tumors

Cancer Informatics ◽

10.4137/cin.s13980 ◽

2014 ◽

Vol 13s4 ◽

pp. CIN.S13980 ◽

Cited By ~ 8

Author(s):

Eugene Andrέs Houseman ◽

Tan A. Ince

Keyword(s):

Breast Cancer ◽

Dna Methylation ◽

Breast Tumors ◽

Cell Types ◽

Normal Breast ◽

Cancer Classification ◽

Methylation Data ◽

Cell Type ◽

Phylogenetic Classification ◽

Breast Cancer Classification

Historically, breast cancer classification has relied on prognostic subtypes. Thus, unlike hematopoietic cancers, breast tumor classification lacks phylogenetic rationale. The feasibility of phylogenetic classification of breast tumors has recently been demonstrated based on estrogen receptor (ER), androgen receptor (AR), vitamin D receptor (VDR) and Keratin 5 expression. Four hormonal states (HR0–3) comprising 11 cellular subtypes of breast cells have been proposed. This classification scheme has been shown to have relevance to clinical prognosis. We examine the implications of such phylogenetic classification on DNA methylation of both breast tumors and normal breast tissues by applying recently developed deconvolution algorithms to three DNA methylation data sets archived on Gene Expression Omnibus. We propose that breast tumors arising from a particular cell-of-origin essentially magnify the epigenetic state of their original cell type. We demonstrate that DNA methylation of tumors manifests patterns consistent with cell-specific epigenetic states, that these states correspond roughly to previously posited normal breast cell types, and that estimates of proportions of the underlying cell types are predictive of tumor phenotypes. Taken together, these findings suggest that the epigenetics of breast tumors is ultimately based on the underlying phylogeny of normal breast tissue.

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Expression of BCL2L12, a new member of apoptosis-related genes, in breast tumors

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613411 ◽

2003 ◽

Vol 89 (06) ◽

pp. 1081-1088 ◽

Cited By ~ 29

Author(s):

Maroulio Talieri ◽

Eleftherios Diamandis ◽

Nikos Katsaros ◽

Dimitrios Gourgiotis ◽

Andreas Scorilas

Keyword(s):

Breast Cancer ◽

Cell Death ◽

Cox Regression ◽

Breast Tumors ◽

Cell Types ◽

Research Society ◽

Genetic Alterations ◽

Cox Regression Analysis ◽

Reverse Transcription Pcr ◽

Lower Stage

SummaryApoptosis, a normal physiological form of cell death, is critically involved in the regulation of cellular homeostasis. If the delicate balance between cell death and cell proliferation is altered by a defect in the normal regulation of apoptosis signaling, a cell population is able to survive and accumulate, thereby favoring the acquisition of further genetic alterations and promoting tumorigenesis. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in the responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-XL has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Recently, a new member of the Bcl-2 family, BCL2L12, was cloned. The BCL2L12 gene is constitutively expressed in many tissues, suggesting that the encoded protein serves an important function in different cell types. In the present study, the expression of BCL2L12 gene was analyzed by reverse transcription-PCR (PT-PCR) in 70 breast cancer tissues. Our results indicate that BCL2L12 positive breast tumors are mainly of lower stage (I/II) or grade (I/II) (p=0.02 or p=0.04 respectively). Cox regression analysis revealed that BCL2L12 expression is positively related to disease-free (DFS) and overall survival (OS) at both univariate and multivariate analysis (p=0.021, p=0.029, p=0.032, p=0.044 respectively). Kaplan-Meier survival curves also demonstrated that patients with BCL2L12-positive tumors have significantly longer DFS and OS (p=0.002 and p<0.001 respectively). BCL2L12 expression may be regarded as a new independent favorable prognostic marker for breast cancer.Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

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Localization of migraine susceptibility genes in human brain by single-cell RNA sequencing

Cephalalgia ◽

10.1177/0333102418762476 ◽

2018 ◽

Vol 38 (13) ◽

pp. 1976-1983 ◽

Cited By ~ 5

Author(s):

William Renthal

Keyword(s):

Human Brain ◽

Single Cell ◽

Rna Sequencing ◽

Expression Profiles ◽

Cell Types ◽

Susceptibility Genes ◽

Brain Cell ◽

Cell Type ◽

Single Cell Rna Sequencing ◽

Brain Cell Types

Background Migraine is a debilitating disorder characterized by severe headaches and associated neurological symptoms. A key challenge to understanding migraine has been the cellular complexity of the human brain and the multiple cell types implicated in its pathophysiology. The present study leverages recent advances in single-cell transcriptomics to localize the specific human brain cell types in which putative migraine susceptibility genes are expressed. Methods The cell-type specific expression of both familial and common migraine-associated genes was determined bioinformatically using data from 2,039 individual human brain cells across two published single-cell RNA sequencing datasets. Enrichment of migraine-associated genes was determined for each brain cell type. Results Analysis of single-brain cell RNA sequencing data from five major subtypes of cells in the human cortex (neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells) indicates that over 40% of known migraine-associated genes are enriched in the expression profiles of a specific brain cell type. Further analysis of neuronal migraine-associated genes demonstrated that approximately 70% were significantly enriched in inhibitory neurons and 30% in excitatory neurons. Conclusions This study takes the next step in understanding the human brain cell types in which putative migraine susceptibility genes are expressed. Both familial and common migraine may arise from dysfunction of discrete cell types within the neurovascular unit, and localization of the affected cell type(s) in an individual patient may provide insight into to their susceptibility to migraine.

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Single-cell RNA sequencing reveals cell type- and artery type-specific vascular remodelling in male spontaneously hypertensive rats

Cardiovascular Research ◽

10.1093/cvr/cvaa164 ◽

2020 ◽

Cited By ~ 1

Author(s):

Jun Cheng ◽

Wenduo Gu ◽

Ting Lan ◽

Jiacheng Deng ◽

Zhichao Ni ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Spontaneously Hypertensive Rats ◽

Cell Types ◽

Vascular Remodelling ◽

Cell Type ◽

Hypertensive Rats ◽

Spontaneously Hypertensive ◽

Single Cell Rna Sequencing ◽

Cell Type Specific

Abstract 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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Estrogen receptor signaling is reprogrammed during breast tumorigenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819155116 ◽

2019 ◽

Vol 116 (23) ◽

pp. 11437-11443 ◽

Cited By ~ 9

Author(s):

David Chi ◽

Hari Singhal ◽

Lewyn Li ◽

Tengfei Xiao ◽

Weihan Liu ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Mammary Gland ◽

Epithelial Cells ◽

Breast Tumors ◽

Normal Mammary Gland ◽

Breast Tumorigenesis ◽

Effective Prevention ◽

Significant Difference ◽

Estrogen Stimulation

Limited knowledge of the changes in estrogen receptor (ER) signaling during the transformation of the normal mammary gland to breast cancer hinders the development of effective prevention and treatment strategies. Differences in estrogen signaling between normal human primary breast epithelial cells and primary breast tumors obtained immediately following surgical excision were explored. Transcriptional profiling of normal ER+ mature luminal mammary epithelial cells and ER+ breast tumors revealed significant difference in the response to estrogen stimulation. Consistent with these differences in gene expression, the normal and tumor ER cistromes were distinct and sufficient to segregate normal breast tissues from breast tumors. The selective enrichment of the DNA binding motif GRHL2 in the breast cancer-specific ER cistrome suggests that it may play a role in the differential function of ER in breast cancer. Depletion of GRHL2 resulted in altered ER binding and differential transcriptional responses to estrogen stimulation. Furthermore, GRHL2 was demonstrated to be essential for estrogen-stimulated proliferation of ER+ breast cancer cells. DLC1 was also identified as an estrogen-induced tumor suppressor in the normal mammary gland with decreased expression in breast cancer. In clinical cohorts, loss of DLC1 and gain of GRHL2 expression are associated with ER+ breast cancer and are independently predictive for worse survival. This study suggests that normal ER signaling is lost and tumor-specific ER signaling is gained during breast tumorigenesis. Unraveling these changes in ER signaling during breast cancer progression should aid the development of more effective prevention strategies and targeted therapeutics.

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CHETAH: a selective, hierarchical cell type identification method for single-cell RNA sequencing

Nucleic Acids Research ◽

10.1093/nar/gkz543 ◽

2019 ◽

Vol 47 (16) ◽

pp. e95-e95 ◽

Cited By ~ 30

Author(s):

Jurrian K de Kanter ◽

Philip Lijnzaad ◽

Tito Candelli ◽

Thanasis Margaritis ◽

Frank C P Holstege

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Reference Data ◽

Classification Tree ◽

Cell Types ◽

Biological Information ◽

Identification Algorithm ◽

Intermediate Cell ◽

Cell Type ◽

Single Cell Rna Sequencing

Abstract Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH’s accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.

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