scholarly journals Resolving Tumour Clonal Heterogeneity and Spatial Complexity using Nuclear Tandem Epitope Protein (nTEP) Barcoding

2018 ◽  
Author(s):  
Michael B. Gill ◽  
Simon Koplev ◽  
Anne C. Machel ◽  
Martin L. Miller
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Tumour Microenvironment ◽  
Cellular Level ◽  
Single Cell Level ◽  
Cellular Interactions ◽  
Cell Level ◽  
Cell Clones ◽  
Mouse Tumour ◽  
Novel Protein

ABSTRACTTumours are composed of an array of unique cancer cell clones along with many non-tumour cells such as immune cells, fibroblasts and endothelial cells, which make up the complex tumour microenvironment. To better understand the co-evolution of tumour clones and cells of the tumour microenvironment, we require tools to spatially resolve heterotypic cellular interactions at the single cell level. We present a novel protein-based barcoding technology termed nuclear tandem epitope protein (nTEP) barcoding, which can be designed to combinatorially encode and track dozens to hundreds of tumour clones in their spatial context within complex cellular mixtures using multiplexed antibody-based imaging. Here we provide proof-of-principle of nTEP barcoding and develop the technology, which relies on lentiviral - based stable expression of a nuclear-localised fluorophore that contains unique combinations of protein epitope tags that can be decoded by a limited set of antibodies. By generating a series of cell lines expressing unique nTEP barcodes, we were able to robustly identify and spatially deconvolve specific clones present within highly complex cell mixtures at the single cell level using state-of-the-art iterative indirect immunofluorescence imaging (4i). We define the utility of nTEP-barcoding as a powerful tool for visualising and resolving tumour heterogeneity at the cellular level, and envision its usage in mouse tumour models for understanding how tumour clones modulate and interact with stromal- and immune cells in cancer.

scholarly journals Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Matthew Ryan Sullivan ◽  
Giovanni Stefano Ugolini ◽  
Saheli Sarkar ◽  
Wenjing Kang ◽  
Evan Carlton Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
Single Cell ◽  
Checkpoint Inhibitors ◽  
Cell Killing ◽  
Calcium Flux ◽  
Single Cell Level ◽  
Cellular Interactions ◽  
Cell Level ◽  
The Impact

AbstractThe inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8+ T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8+ T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8+ T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations of CD8+ T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8+ T cells.

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

2021 ◽  
Author(s):  
Wilson McKerrow ◽  
Shane A. Evans ◽  
Azucena Rocha ◽  
John Sedivy ◽  
Nicola Neretti ◽  
...  
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Early Development ◽  
Immune Cells ◽  
Copy Number ◽  
Single Cells ◽  
Single Cell Level ◽  
Rna Seq ◽  
Cell Level ◽  
Mouse Hippocampus

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.

scholarly journals Mass Cytometry Study on the Heterogeneity in Cellular Association and Cytotoxicity of Silver Nanoparticles in Human Immune Cells

2019 ◽  
Author(s):  
My Kieu Ha ◽  
Jang-Sik Choi ◽  
Zayakhuu Gerelkhuu ◽  
Sook Jin Kwon ◽  
Jaewoo Song ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Adverse Effects ◽  
Single Cell ◽  
Dose Response ◽  
Immune Cells ◽  
Intracellular Signaling ◽  
Single Cell Level ◽  
Mass Cytometry ◽  
Cell Level ◽  
Human Immune

AbstractThere have been many reports about the adverse effects of nanoparticles (NPs) on the environment and human health. Conventional toxicity assessments of NPs frequently assume uniform distribution of monodisperse NPs in homogeneous cell populations, and provide information on the relationships between the administered dose of NPs and cellular responses averaged for a large number of cells. They may have limitations in describing the wide heterogeneity of cell-NP interactions, caused by cell-to-cell and NP-to-NP variances. To achieve more detailed insight into the heterogeneity of cell-NP interactions, it is essential to understand the cellular association and adverse effects of NPs at single-cell level. In this study, we applied mass cytometry to investigate the interactions between silver nanoparticles (AgNPs) and primary human immune cells. High dimensionality of mass cytometry allowed us to identify various immune cell types and observe the cellular association and toxicity of AgNPs in each population. Our findings showed that AgNPs had higher affinity with phagocytic cells like monocytes and dendritic cells and caused more severe toxic effects than with T cells, B cells and NK cells. Multi-element detection capability of mass cytometry also enabled us to simultaneously monitor cellular AgNP dose and intracellular signaling of individual cells, and subsequently investigate the dose-response relationships of each immune population at single-cell level, which are often hidden in conventional toxicity assays at bulk-cell level. Our study will assist future development of single-cell dose-response models for various NPs and will provide key information for the safe use of nanomaterials for biomedical applications.

scholarly journals Enhancing Our Understanding of Plant Cell-to-Cell Interactions Using Single-Cell Omics

2021 ◽  
Vol 12 ◽  
Author(s):  
Sandra Thibivilliers ◽  
Marc Libault
Keyword(s):  
Single Cell ◽  
Hair Cells ◽  
Plant Cells ◽  
Single Cell Level ◽  
Cellular Interactions ◽  
Cell Level ◽  
Dynamic Regulation ◽  
Omics Technologies ◽  
Spatial Profiling ◽  
Root Hair Cells

Plants are composed of cells that physically interact and constantly adapt to their environment. To reveal the contribution of each plant cells to the biology of the entire organism, their molecular, morphological, and physiological attributes must be quantified and analyzed in the context of the morphology of the plant organs. The emergence of single-cell/nucleus omics technologies now allows plant biologists to access different modalities of individual cells including their epigenome and transcriptome to reveal the unique molecular properties of each cell composing the plant and their dynamic regulation during cell differentiation and in response to their environment. In this manuscript, we provide a perspective regarding the challenges and strategies to collect plant single-cell biological datasets and their analysis in the context of cellular interactions. As an example, we provide an analysis of the transcriptional regulation of the Arabidopsis genes controlling the differentiation of the root hair cells at the single-cell level. We also discuss the perspective of the use of spatial profiling to complement existing plant single-cell omics.

scholarly journals Dynamical transitions of the actomyosin cortex can trigger single cell morphogenesis

2021 ◽  
Author(s):  
Hongkang Zhu ◽  
Roberto Alonso-Matilla ◽  
Zachary A McDargh ◽  
Ben O'Shaughnessy
Keyword(s):  
Single Cell ◽  
Complex Shape ◽  
Tissue Level ◽  
Cellular Level ◽  
Spatial Patterning ◽  
Single Cell Level ◽  
Cell Level ◽  
Complex Shapes ◽  
Contractile Forces ◽  
Shape Changes

Morphogenetic changes driven by actomyosin contractile forces are well-characterized at the tissue level. At the single cell level, shape changes steered by actomyosin contractile forces include mitotic rounding and cytokinetic furrow ingression. In some cases, more complex shape transitions associated with spatial patterning of the cortex were observed. The actomyosin cortex was widely studied using active gel frameworks, and stabilized contractile instabilities were shown to generate patterns, but whether complex shapes can emerge from these cortical patterns is not established. Here we show that complex morphogenetic changes at the single cell level can accompany cortical patterns, using a minimal active gel model. For sufficiently low membrane-cortex drag, an initially homogeneous cortex spontaneously develops stripes associated with stable furrows, similar to furrowing observed in cells. Our work suggests that controlled cortical instability can trigger morphogenesis at the cellular level.

scholarly journals Advances in single cell technologies in immunology

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 226-236
Author(s):  
Jane Ru Choi
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Immune Cell ◽  
Single Cell Level ◽  
Future Perspectives ◽  
Biological Mechanisms ◽  
Cell Level ◽  
Cell Responses ◽  
Physiological Processes ◽  
Cell Technologies

The immune system is composed of heterogeneous populations of immune cells that regulate physiological processes and protect organisms against diseases. Single cell technologies have been used to assess immune cell responses at the single cell level, which are crucial for identifying the causes of diseases and elucidating underlying biological mechanisms to facilitate medical therapy. In the present review we first discuss the most recent advances in the development of single cell technologies to investigate cell signaling, cell–cell interactions and cell migration. Each technology's advantages and limitations and its applications in immunology are subsequently reviewed. The latest progress toward commercialization, the remaining challenges and future perspectives for single cell technologies in immunology are also briefly discussed.

Energetics at the Single Cell Level

Physiology ◽  
1989 ◽  
Vol 4 (3) ◽  
pp. 91-93
Author(s):  
WJ van der Laarse ◽  
G Elzinga ◽  
RC Woledge
Keyword(s):  
Single Cell ◽  
Energy Flow ◽  
Cellular Level ◽  
Single Cell Level ◽  
Cell Level

Recently developed techniques allow the study of energy flow at the cellular level and show in new detail how cells are individually adapted for their particular energetic roles.

Abstract 439: High Dimensional Single-Cell Immune Contexture of Human Atherosclerotic Plaques and Blood

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Adeeb Rahman ◽  
Aleksey Chudnovskiy ◽  
El-ad David Amir ◽  
Seunghee Kim-Schulze ◽  
Jennifer R Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
High Dimensional ◽  
Specific Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Immune Contexture

Atherosclerosis is a disease characterized by immune infiltration of the arterial wall in response to tissue damage and systemic inflammation. In the era of precision medicine, is essential to gain insights on immune contexture of atherosclerotic tissue taking into account disease-specific cell variation in patients. We applied high-dimensional technologies for the analysis of multiple parameters at the single-cell level in clinical samples of patients undergoing carotid endatherectomy (CEA, n=15). Using time-of-flight mass-cytometry (CyTOF), we simultaneously analyzed 32 parameters at the single-cell level in peripheral blood mononuclear cells (PBMCs) and atherosclerotic-tissue associated immune cells of the same patient. Using viSNE, we mapped single-cell heterogeneity into two dimensions to discriminate PBMCs and tissue-associated CD45+ immune cells. Next, we employed Phenograph to cluster cells into phenotypically related populations, which were annotated based on canonical marker expression patterns. We identified several major immune subsets including two subsets of macrophages (CD163 low and CD163 high ), monocytes, dendritic cells (DCs), B and T cells. The most prevalent CD45+ cells identified in atherosclerotic tissue were CD4 + (25.8%) and CD8 + (25.2%) T cells, macrophages (12.8%), monocytes (7.7%) and B (2.1%) cells. Using a regression analysis similar to that employed by CITRUS, we determined that macrophages and a subset of CD8 T cells characterized by low expression of CD127 were selectively enriched in tissue vs. blood. Multiplexed immunohistochemistry confirmed that T cells comprised a major portion of the CD45+ cells in atherosclerotic tissue, even more abundant than macrophages. This study of deep phenotyping across-atherosclerotic tissue and blood demonstrate a significant T cell tissue infiltration of a specific subset of CD8 T cells. This suggests that adaptive T cell immunity plays a critical role in advanced atherosclerosis. The extension of this systems biology analysis pipeline to larger datasets can improve our understanding of the core mechanisms of chronic inflammation in atherosclerosis.

Sign in / Sign up

Export Citation Format

Share Document