LINE-1 Retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5’-single cell RNA-Seq

AbstractLINE-1 retrotransposons are known to be expressed in early development, in tumors and in the germline. Less is known about LINE-1 expression at the single cell level, especially outside the context of cancer. Because LINE-1 elements are present at a high copy number, many transcripts that are not driven by the LINE-1 promoter nevertheless terminate at the LINE-1 3’ UTR. Thus, 3’ targeted single cell RNA-seq datasets are not appropriate for studying LINE-1. However, 5’ targeted single cell datasets provide an opportunity to analyze LINE-1 expression at the single cell level. Most LINE-1 copies are 5’ truncated, and a transcript that contains the LINE-1 5’ UTR as its 5’ end is likely to have been transcribed from its promoter. We developed a method, L1-sc (LINE-1 expression for single cells), to quantify LINE-1 expression in 5’ targeted 10x genomics single cell RNA-seq datasets. Our method confirms that LINE-1 expression is high in cancer cells, but low or absent from immune cells. We also find that LINE-1 expression is elevated in epithelial compared to immune cells outside of the context of cancer and that it is also elevated in neurons compared to glia in the mouse hippocampus.

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Efficient analysis of a small number of cancer cells at the single-cell level using an electroactive double-well array

Lab on a Chip ◽

10.1039/c6lc00241b ◽

2016 ◽

Vol 16 (13) ◽

pp. 2440-2449 ◽

Cited By ~ 26

Author(s):

Soo Hyeon Kim ◽

Teruo Fujii

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Single Cells ◽

Capture Efficiency ◽

Single Cell Level ◽

Cell Capture ◽

Cell Level ◽

Cell Trapping ◽

Highly Efficient ◽

Single Cell Trapping

The electroactive double well-array consists of trap-wells for highly efficient single-cell trapping using dielectrophoresis (cell capture efficiency of 96 ± 3%) and reaction-wells that confine cell lysates for analysis of intracellular materials from single cells.

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Centrifugal Microfluidics Traps for Parallel Isolation and Imaging of Single Cells

Micromachines ◽

10.3390/mi11020149 ◽

2020 ◽

Vol 11 (2) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Adam Snider ◽

Ileana Pirozzi ◽

Anubhav Tripathi

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Single Cells ◽

Unmet Need ◽

Centrifugal Microfluidics ◽

Cancer Tissue ◽

Single Cell Level ◽

Cell Level ◽

Tissue Samples ◽

Potential Applications

Analysis at the single cell level has becoming an increasingly important procedure to diagnose cancer tissue biopsies. These tissue samples are often heterogeneous and consist of 1000–15,000 cells. We study the use of centrifugal microfluidics to isolate single cells into micro chambers. We describe the optimization of our microfluidics flow device, characterize its performance using both polystyrene beads as a cell analogue and MCF-7 breast cancer cells, and discuss potential applications for the device. Our results show rapid isolation of ~2000 single cell aliquots in ~20 min. We were able to occupy 65% of available chambers with singly occupied cancer cells, and observed capture efficiencies as high as 80% using input samples ranging from 2000 to 15,000 cells in 20 min. We believe our device is a valuable research tool that addresses the unmet need for massively parallel single cell level analysis of cell populations.

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High-throughput imaging of mRNA at the single-cell level in human primary immune cells

10.1101/2020.11.10.377283 ◽

2020 ◽

Author(s):

Manasi Gadkari ◽

Jing Sun ◽

Adrian Carcamo ◽

Hugh Alessi ◽

Zonghui Hu ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Immune Cells ◽

Single Cells ◽

Transcript Abundance ◽

Hybridization Chain Reaction ◽

Single Cell Level ◽

Cell Level ◽

Experimental Conditions ◽

General Applicability

AbstractMeasurement of gene expression at the single-cell level has led to important advances in the study of transcriptional regulation programs in healthy and disease states. In particular, single-cell gene expression approaches have shed light on the high level of transcriptional heterogeneity of individual cells, both at baseline and in response to experimental or environmental perturbations. We have developed a method for High-Content Imaging (HCI)-based quantification of transcript abundance at the single-cell level in primary human immune cells and have validated its performance under multiple experimental conditions to demonstrate its general applicability. This method, which we abbreviate as hcHCR, combines the high sensitivity of the hybridization chain reaction (HCR) for the visualization of mRNA molecules in single cells, with the speed, scalability, and technical reproducibility of HCI. We first tested eight microscopy-compatible attachment substrates for short-term culture of primary human B cells, T cells, monocytes, or neutrophils. We then miniaturized HCR in a 384-well format and documented the ability of the method to detect increased or decreased transcript abundance at the single-cell level in thousands of cells for each experimental condition by HCI. Furthermore, we demonstrated the feasibility of multiplexing gene expression measurements by simultaneously assaying the abundance of two transcripts per cell, both at baseline and in response to an experimental stimulus. Finally, we tested the robustness of the assay to technical and biological variation. We anticipate that hcHCR will be a suitable and cost-effective assay for low- to medium-throughput chemical, genetic or functional genomic screens in primary human cells, with the possibility of performing personalized screens or screens on cells obtained from patients with a specific disease.

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SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

Science Advances ◽

10.1126/sciadv.abe3610 ◽

2021 ◽

Vol 7 (8) ◽

pp. eabe3610

Author(s):

Conor J. Kearney ◽

Stephin J. Vervoort ◽

Kelly M. Ramsbottom ◽

Izabela Todorovski ◽

Emily J. Lelliott ◽

...

Keyword(s):

Single Cell ◽

Posttranslational Modification ◽

Cellular Differentiation ◽

Single Cells ◽

Simultaneous Detection ◽

Surface Protein ◽

Rna Seq ◽

Cell Level ◽

Simultaneous Integration ◽

Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

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Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽

10.3390/cells11020285 ◽

2022 ◽

Vol 11 (2) ◽

pp. 285

Author(s):

Eszter Széles ◽

Krisztina Nagy ◽

Ágnes Ábrahám ◽

Sándor Kovács ◽

Anna Podmaniczki ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Single Cell ◽

Single Cells ◽

Fluorescence Induction ◽

Photochemical Quenching ◽

Support Surface ◽

Single Cell Level ◽

Microfluidic Platforms ◽

Cell Level ◽

Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

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Characterizing Intercellular Communication of Pan-Cancer Reveals SPP1+ Tumor-Associated Macrophage Expanded in Hypoxia and Promoting Cancer Malignancy Through Single-Cell RNA-Seq Data

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.749210 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jinfen Wei ◽

Zixi Chen ◽

Meiling Hu ◽

Ziqing He ◽

Dawei Jiang ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Single Cells ◽

Matrix Remodeling ◽

Rna Seq ◽

Mesenchymal Transition ◽

Tumor Associated Macrophage ◽

Cancer Types

Hypoxia is a characteristic of tumor microenvironment (TME) and is a major contributor to tumor progression. Yet, subtype identification of tumor-associated non-malignant cells at single-cell resolution and how they influence cancer progression under hypoxia TME remain largely unexplored. Here, we used RNA-seq data of 424,194 single cells from 108 patients to identify the subtypes of cancer cells, stromal cells, and immune cells; to evaluate their hypoxia score; and also to uncover potential interaction signals between these cells in vivo across six cancer types. We identified SPP1+ tumor-associated macrophage (TAM) subpopulation potentially enhanced epithelial–mesenchymal transition (EMT) by interaction with cancer cells through paracrine pattern. We prioritized SPP1 as a TAM-secreted factor to act on cancer cells and found a significant enhanced migration phenotype and invasion ability in A549 lung cancer cells induced by recombinant protein SPP1. Besides, prognostic analysis indicated that a higher expression of SPP1 was found to be related to worse clinical outcome in six cancer types. SPP1 expression was higher in hypoxia-high macrophages based on single-cell data, which was further validated by an in vitro experiment that SPP1 was upregulated in macrophages under hypoxia-cultured compared with normoxic conditions. Additionally, a differential analysis demonstrated that hypoxia potentially influences extracellular matrix remodeling, glycolysis, and interleukin-10 signal activation in various cancer types. Our work illuminates the clearer underlying mechanism in the intricate interaction between different cell subtypes within hypoxia TME and proposes the guidelines for the development of therapeutic targets specifically for patients with high proportion of SPP1+ TAMs in hypoxic lesions.

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High-resolution label-free 3D mapping of extracellular pH of single living cells

Nature Communications ◽

10.1038/s41467-019-13535-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Yanjun Zhang ◽

Yasufumi Takahashi ◽

Sung Pil Hong ◽

Fengjie Liu ◽

Joanna Bednarska ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Extracellular Ph ◽

Single Cell Level ◽

Ph Sensing ◽

Label Free ◽

Cell Level ◽

Spatiotemporal Resolution ◽

Ion Conductance ◽

Scanning Ion Conductance Microscopy

AbstractDynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

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Dissecting CLL through high-dimensional single-cell technologies

Blood ◽

10.1182/blood-2018-09-835389 ◽

2019 ◽

Vol 133 (13) ◽

pp. 1446-1456

Author(s):

Satyen H. Gohil ◽

Catherine J. Wu

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Single Cell ◽

Cancer Cells ◽

Lymphocytic Leukemia ◽

Response To Treatment ◽

High Dimensional ◽

Single Cell Level ◽

Disease Course ◽

Cell Level ◽

New Techniques

Abstract We now have the potential to undertake detailed analysis of the inner workings of thousands of cancer cells, one cell at a time, through the emergence of a range of techniques that probe the genome, transcriptome, and proteome combined with the development of bioinformatics pipelines that enable their interpretation. This provides an unprecedented opportunity to better understand the heterogeneity of chronic lymphocytic leukemia and how mutations, activation states, and protein expression at the single-cell level have an impact on disease course, response to treatment, and outcomes. Herein, we review the emerging application of these new techniques to chronic lymphocytic leukemia and examine the insights already attained through this transformative technology.

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Probing the Interaction Forces of Prostate Cancer Cells with Collagen I and Bone Marrow Derived Stem Cells on the Single Cell Level

PLoS ONE ◽

10.1371/journal.pone.0057706 ◽

2013 ◽

Vol 8 (3) ◽

pp. e57706 ◽

Cited By ~ 14

Author(s):

Ediz Sariisik ◽

Denitsa Docheva ◽

Daniela Padula ◽

Cvetan Popov ◽

Jan Opfer ◽

...

Keyword(s):

Prostate Cancer ◽

Stem Cells ◽

Bone Marrow ◽

Single Cell ◽

Cancer Cells ◽

Collagen I ◽

Single Cell Level ◽

Prostate Cancer Cells ◽

Cell Level ◽

Interaction Forces

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Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Applied and Environmental Microbiology ◽

10.1128/aem.02625-08 ◽

2009 ◽

Vol 75 (13) ◽

pp. 4550-4556 ◽

Cited By ~ 27

Author(s):

Vicky G. Kastbjerg ◽

Dennis S. Nielsen ◽

Nils Arneborg ◽

Lone Gram

Keyword(s):

Listeria Monocytogenes ◽

Single Cell ◽

Intracellular Ph ◽

Bacterial Population ◽

Single Cells ◽

Viable Cell ◽

Population Subdivision ◽

Single Cell Level ◽

Cell Level ◽

Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

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