Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics

Rivello, Francesca; Buijtenen, Erik van; Matuła, Kinga; Buggenum, Jessie A.G.L. van; Vink, Paul; Eenennaam, Hans van; Mulder, Klaas W.; Huck, Wilhelm T. S.

doi:10.1016/j.crmeth.2021.100070

Cited by 24 publications

(26 citation statements)

References 22 publications

(29 reference statements)

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“…To quantify signaling pathway activity (i.e. phosphorylation levels), surface markers, and mRNA expression on single-cell level, we developed a strategy based on CITE-seq ( 16 ), Immuno-Detection by Sequencing (ID-seq) ( 18, 19 ), RNA and Immuno-detection (RAID) ( 22 ) and droplet-based QuRIE-seq ( 20 ). To enable immunostaining and mRNA analysis on live ASCs for CITE-seq-like sample-preparation and immunostaining on fixed and permeabilized ASCs for intracellular immuno-detection by sequencing, we divided the ASCs from each donor into two portions before staining and sorting for multi-modal single-cell analysis (Fig 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, in IgG we detected active IL-6 signaling and a NF-κB signature. This signaling axis, in concert with the BLIMP1-XBP1 axis, has been described to induce mTOR activity, and antibody production through regulating the unfolded protein response ( 20, 54 ). Taken together with the phenotypic markers (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…Single-cell sequencing technologies have revolutionized our insight into the molecular phenotyping of many cell types of the human body. The combined quantification of mRNA and surface protein markers allows for additional (sub-)classification of cells ( 16, 17 ), while immune-detection by sequencing (ID-seq) ( 18, 19 ) and droplet-based QuRIE-seq ( 20 ) allows quantification of intracellular phospho-proteins. These methods rely on the reversible fixation of cells and immunostaining with DNA-tagged antibodies, enabling RNA sequencing and quantification of (phospho-)proteins in a single assay.…”

Section: Introductionmentioning

confidence: 99%

“…These methods rely on the reversible fixation of cells and immunostaining with DNA-tagged antibodies, enabling RNA sequencing and quantification of (phospho-)proteins in a single assay. Unfortunately, our fixation-based methods did not result in high-quality mRNA libraries in primary immune-cells ( 20 ). To circumvent this technical challenge, we combined multi-modal data from fixed and non-fixed cells.…”

Section: Introductionmentioning

confidence: 99%

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Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells

Buijtenen

Janssen²,

Vink³

et al. 2022

Preprint

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Antibody-secreting cells (ASCs) secrete IgM, IgA, or IgG antibodies and are key components of humoral immunity; however, little is known about unique characteristics of the Ig-classes due to limited availability of material and challenges to quantify many intracellular molecular modalities at a single-cell resolution. We combined a method to in vitro differentiate peripheral B-cells into ASCs with integrated multi-omic single-cell sequencing technologies to quantify subclass-specific hallmark surface markers, transcriptional profiles and signaling transduction pathway components. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors and IL-2, IL-6, JAK/STAT and mTOR signaling activity across Ig-subclasses. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a complex sample of in vitro differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells

Buijtenen

Janssen²,

Vink³

et al. 2022

Preprint

Self Cite

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“…Additionally, there are no high-throughput single-cell techniques capable of capturing both changes in phosphorylation levels and gene expression patterns [ 67 , 68 ]. Rivello et al presented a quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a high-throughput platform for quantifying intra- and extracellular (phospho)proteins simultaneously, and the transcriptome within thousands of single cells.…”

Section: Single-cell Proteomics In Cancermentioning

confidence: 99%

Single-Cell Proteomics: The Critical Role of Nanotechnology

Arias-Hidalgo

Juanes-Velasco

Landeira-Viñuela

et al. 2022

IJMS

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In single-cell analysis, biological variability can be attributed to individual cells, their specific state, and the ability to respond to external stimuli, which are determined by protein abundance and their relative alterations. Mass spectrometry (MS)-based proteomics (e.g., SCoPE-MS and SCoPE2) can be used as a non-targeted method to detect molecules across hundreds of individual cells. To achieve high-throughput investigation, novel approaches in Single-Cell Proteomics (SCP) are needed to identify and quantify proteins as accurately as possible. Controlling sample preparation prior to LC-MS analysis is critical, as it influences sensitivity, robustness, and reproducibility. Several nanotechnological approaches have been developed for the removal of cellular debris, salts, and detergents, and to facilitate systematic sample processing at the nano- and microfluidic scale. In addition, nanotechnology has enabled high-throughput proteomics analysis, which have required the improvement of software tools, such as DART-ID or DO-MS, which are also fundamental for addressing key biological questions. Single-cell proteomics has many applications in nanomedicine and biomedical research, including advanced cancer immunotherapies or biomarker characterization, among others; and novel methods allow the quantification of more than a thousand proteins while analyzing hundreds of single cells.

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Exploring New Dimensions of Tumor Heterogeneity: The Application of Single Cell Analysis to Organoid‐Based 3D In Vitro Models

Landon‐Brace,

Li,

McGuigan

2023

Adv Healthcare Materials

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Modelling the heterogeneity of the tumor microenvironment (TME) in vitro is essential to investigating fundamental cancer biology and developing novel treatment strategies that holistically address the factors affecting tumor progression and therapeutic response. Thus, the development of new tools for both in vitro modelling, such as patient‐derived organoids (PDOs) and complex 3D in vitro models, and single cell omics analysis, such as single‐cell RNA‐sequencing, represents a new frontier for investigating tumor heterogeneity. Specifically, the integration of PDO‐based 3D in vitro models and single cell analysis offers a unique opportunity to explore the intersecting effects of interpatient, microenvironmental, and tumor cell heterogeneity on cell phenotypes in the TME. In this review, we discuss the current use of PDOs in complex 3D in vitro models of the TME and highlight emerging directions in the development of these models. Next, we examine work that has successfully applied single cell analysis to PDO‐based models and identify important experimental considerations for this approach. Finally, we highlight open questions that may be amenable to exploration using the integration of PDO‐based models and single cell analysis. Ultimately, such investigations may facilitate the identification of novel therapeutic targets for cancer that address the significant influence of tumor‐TME interactions.This article is protected by copyright. All rights reserved

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Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics

Cited by 24 publications

References 22 publications

Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells

Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells

Single-Cell Proteomics: The Critical Role of Nanotechnology

Exploring New Dimensions of Tumor Heterogeneity: The Application of Single Cell Analysis to Organoid‐Based 3D In Vitro Models

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