March Literature Blitz
Time：2022-04-07 17:43:34
March Top 10 Publications - Advances in Single Cell Sequencing
Our monthly single cell Literature Blitz column is here again. What new achievements in single cell sequencing has March brought us? Here is our selection of top publications featuring new advances and applications of single cell sequencing in understanding and treatment of spinal cord injury, heart failure, leukemia, COVID-19, cancer metastasis, ageing, and development.
What are your March top choices?
[1] The Role of Microglia in Spinal Cord Injury
[2] Drug Repurposing in Heart Failure
[3] Drug Resistance Landscape in Leukemia
[5] LIBRA-seq: Neutralizing Antibody (nAb) Discovery
[6] Spatial Transcriptomics in Cancer Metastasis
[7] New method: CellenONE and ICELL8 combination
[9] Zebrafish Sensory Hair Regeneration
The Role of Microglia in Spinal Cord Injury [↑]
[1] Tansley et al. Single-cell RNA sequencing reveals time- and sex-specific responses of mouse spinal cord microglia to peripheral nerve injury and links ApoE to chronic pain. Nature Communications, 2022. [PubMed]
The role of microglia in neuropathic pain is known to be sex dependent. Microglia proliferation and morphological changes in response to peripheral nerve injury are present in both sexes. However, a functional role of microglia as critical drivers of neuropathic pain is observed in male but not in female animals.
Tansley et al. used single-cell RNA-sequencing to uncover the microglia subpopulations or states which contribute to different stages of pain development and maintenance. They showed that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype and demonstrated increased proliferation of microglia in male as compared to female mice. Also, upregulation of Apoe, a gene that authors showed is strongly associated with human chronic pain conditions, might represent a central mechanism in this switch and its further examination can generate important insights into the microglia-dependent mechanisms of neuropathic pain.
Figure 1: Spinal cord microglia subpopulations. (e) UMAP plot reveals that microglia in the mouse spinal cord in all conditions are present in 11 distinct clusters. Inset shows proportion of cells in each cluster in naive mice. (f) UMAP plot showing the expression (log-normalized counts) of canonical microglial genes. (g) Expression of a top unique gene in the indicated cluster and its UMAP plot are shown.
Drug Repurposing in Heart Failure [↑]
[2] Wan et al. Single cell study of cellular diversity and mutual communication in chronic heart failure and drug repositioning. Genomics, 2022. [PubMed]
Heart failure (HF) occurs when the heart muscle doesn't pump blood as well as it should. After the infarction occurs, the injured heart often goes through a series of delicate and coordinated events to compensate for its impaired function. Many non-cardiomyocytes (non-CMs), act as an essential functional element of the heart, inevitably undergoing a series of changes in the composition and biological functions of chain molecules in response to various stimuli.
Wan et al. aimed to characterize the cellular landscape of non-CMs in mice with chronic heart failure by using single-cell RNA sequencing and provide potential therapeutic directions. They showed that fibroblasts and endothelial cells are the driving hubs in chronic heart failure. Combining single cell data with Concept Mapping (CMap), a form of machine learning, it was possible to repurpose already available drugs targeting different cells. Histone DeAaCetylation (HDAC) and Heat Shock Protein (HSP) inhibitors were identified as new potential anti-heart failure drugs.
Figure 2: Cellular atlas of non-CMs in mouse heart. Two-dimensional, t-distributed stochastic neighbor embedding (t-SNE) projections of 17, 853 cardiac cells derived from scRNA-seq data in Sham and HF groups.
Drug Resistance Landscape in Leukemia. [↑]
[3] Thompson et al. Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors. Blood Advances, 2022. [PubMed]
Targeted Agents (TAs) are commonly used to treat Chronic Lymphocytic Leukemia (CLL). These include Bruton Tyrosine Kinase inhibitors (BTKi) and the selective B-cell Lymphoma 2 (BCL2) inhibitor venetoclax, however, despite their substantial efficacy, emergent resistance is a significant cause of treatment failure. The treatment’s selective pressure on the cell landscape results in mutations of the drug target’s binding site or an activating mutation in alternative pathways.
Thompson et al. investigated the clonal structure and evolution of resistance in patients with CLL whose disease harbors multiple resistance mutations to either a single or sequential TAs (BTK, PLCG2, or BCL2). Clone analysis showed that individual nucleotide changes arose multiple times independently in some patients, indicating further clonal complexity within these disease cell populations that cannot be detected by bulk sequencing and the possibility of additional variant-identical clones not distinguishable by targeted sequencing. This study highlights the power of single cell sequencing in drug resistance characterization.
Figure 3: Clonal relationships between resistance mutations in CLL (n = 8 patients) and MCL (n = 1 patient) inferred from variant-based analysis of single cell data.
COVID-19 and Autoimmunity [↑]
[4] Jing et al. KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19. Science, 2022. [PubMed]
Most CD8+ T cells are geared toward the control of pathogen-infected or cancerous cells. There has been long-standing evidence in mice that a small subset can also suppress autoimmune responses, expressing Ly49. The Ly49 family of inhibitory C-type lectin-like receptors, which are ubiquitous on natural killer (NK) cells, were identified as unique surface markers for this regulatory CD8+ T cell subset. However, the human counterparts of these cells were never previously characterized.
Jing et al. identified CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs), the functional counterpart of the mouse Ly49 family in humans, as a regulatory CD8+ T cell subset in humans that suppresses pathogenic CD4+ T cells in celiac disease (CeD), and likely other autoimmune disorders and infectious diseases as well. Also, they found that KIR+CD8+ T cells were substantially elevated in many SARS-CoV-2 patients and higher levels correlated with more severe disease, implicating these cells in the autoimmune states during Acute Respiratory Distress Syndrome (ARDS). This study is a good example of how single cell data mining can be applied to expanding and validating novel prognostic markers in autoimmunity and infectious diseases.
Figure 4: Single cell RNA sequencing analysis of KIR+CD8+ T cells in the blood. Examples provided: COVID-19, healthy controls (HC) and Multiple sclerosis (MS)
LIBRA-seq: Neutralizing Antibody (nAb) Discovery [↑]
[5] Shiakolas et al. KIR+CD8+ Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking. Nature Biotechnologies, 2022.[PubMed]
nAb development is generally inefficient and requires time-intensive subsequent validation steps. Antibodies that block the interactions between the SARS-CoV-2 S protein and its host receptor ACE2 are among the most potent nAbs identified to date. Integration of target–ligand blocking with a previously described B cell receptor-sequencing approach (LInking B cell Receptor to Antigen specificity through sequencing - LIBRA-seq) enables the rapid and efficient identification of multiple neutralizing mAbs.
Shiakolas et al. used LIBRA-seq to identify multiple neutralizing antibodies that prevent binding of SARS-CoV-2 S protein to ACE2, showing higher throughput and potential for a more comprehensive coverage of antibody epitopes. This method offers significant advantages for rapid development of therapeutic and preventive countermeasures to infectious diseases like COVID-19.
Figure 5: LIBRA-seq with a SARS-CoV-2 S titration and ligand blocking.
Spatial Transcriptomics in Cancer Metastasis [↑]
[6] Wu et al. Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level. Cancer Discovery, 2022.[PubMed]
Liver metastasis remains a major hurdle to long-lasting survival of patients with colorectal cancer, which can be partly explained by the highly dynamic spreading routes of cancer cells. TME of liver metastasis harbours a highly immunosuppressive phenotype, induces a systemic loss of antigen-specific T lymphocytes, and drives the spread of tumours. It still remains largely unknown how the immune cells spatially orchestrate colorectal cancer liver metastasis (CRLM) progression and whether the metastatic cellular microenvironment differs from the primary ones
Wu et al. implemented spatial transcriptomics for the analysis of the metastatic microenvironment and showed that it underwent a remarkable spatial reprogramming of immunosuppressive cells such as MRC1+ CCL18+ M2-like macrophages. They also developed scMetabolism, a computational pipeline for quantifying single-cell metabolism, and observed that those macrophages harbored enhanced metabolic activity.
Figure 6: Transcriptomics and spatio-temporal analysis of cancer tissue. (A) All main type cells. (B) Subpopulation of myeloid cells and CD8+ T cells. (C) The unsupervised clustering analysis of colorectal cancer and LM.
New method: CellenONE and ICELL8 combination [↑]
[7] Shomroni et al. A novel single‑cell RNA‑sequencing approach and its applicability connecting genotype to phenotype in ageing disease. Scientific Reports, 2022.[PubMed]
One of the crucial steps in performing single cell sequencing is obtaining the single-cell suspension. This step is prone to errors in terms of bias in cell representation, preserving the viability of the cells, or distinguishing between cellular and acellular material. Methods such as CellenONE and ICELL8 do some things well while being limited in other aspects.
Shomroni et al. overcame the limitations of CellenONE and ICELL8 by combining the methods to harvest the strengths of both. Cells preprocessed on CellenONE X1 were further processed with the ICELL8 system to remove artifacts and doublets. The sensitivity was benchmarked by measuring heterogeneity in accelerated ageing phenotypes. The combination also captured more intronic and intergenic regions and uncovered additional unique markers.
Figure 7: Analysis of transcriptional heterogeneity of dermal fibroblast derived from patients using the novel platform. (a) t-SNE of the ICELL8 and CellenONE-ICELL8.
Ageing of Human Skin [↑]
[8] Sole-Boldo et al. Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming. Nature, 2022.[PubMed]
Dermal fibroblasts play a fundamental role in skin architecture, actively participating in cutaneous immune responses, wound healing, and communication with the nervous and vascular system. However, their heterogeneity has not been analyzed systematically yet.
To characterize fibroblast heterogeneity and reveal the functional diversity of dermal fibroblasts, Sole-Boldo et al. implemented single cell sequencing on whole-skin specimens from male donors. Their results defined four main subpopulations that can be spatially localized and show differential secretory, mesenchymal, and pro-inflammatory functional annotations. Also, they showed that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Taken together, this study widened our understanding of human skin aging and its associated phenotypes.
Figure 8: Aging leads to loss of dermal fibroblast priming.
Zebrafish Sensory Hair Regeneration [↑]
[9] Baek et al. Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration. Developmental Cell, 2022.[PubMed]
Humans and mammals in general fail to regenerate mechanosensory hair cells (HCs) in their inner ears. Hearing loss due to HC death is therefore permanent. Zebrafish and mammalian sensory HCs are very similar; however, zebrafish constantly replace dying HCs through proliferation and differentiation of support cells (SCs). Identifying the blueprint of HC regeneration in zebrafish could be used to experimentally trigger regeneration in mammals. Baek et al. investigated when lineage decisions occur and in which cell the underlying genes and pathways are modulated, broadening our understanding of zebrafish lateral line HC regeneration. They found that regeneration depends on three subsequently activated gene modules that can serve as a blueprint to trigger regeneration in mammals.
Figure 9: Spatiotemporal scRNA-seq analysis during lateral line HC regeneration.
Sea Urchin Morphogenesis [↑]
[10] Satoh et al. A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos. Developmental Biology, 2022.[PubMed]
Single cell sequencing is being implemented increasingly more often in animals other than the standard model organisms. Sea urchin embryos provide a model experimental system to explore gene regulatory networks (GRNs) responsible for specification and differentiation of early embryonic cells. Satoh et al. aimed to study cell clusters expressing transcription factor Brachyury in relation to the morphogenesis. They found that this transcription factor is expressed in the invaginating endoderm, and oral ectoderm of the sea urchin, confirming the previous model that the ventral organizer is a property of Brachyury-positive oral ectodermal cells.
Figure 10: Sea urchin single cell sequencing analysis.
References [↑]
[1] Tansley et al. Single-cell RNA sequencing reveals time- and sex-specific responses of mouse spinal cord microglia to peripheral nerve injury and links ApoE to chronic pain. Nature Communications, 2022. [PubMed]
[2] Wan et al. Single cell study of cellular diversity and mutual communication in chronic heart failure and drug repositioning. Genomics, 2022. [PubMed]
[3] Thompson et al. Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors. Blood Advances, 2022. [PubMed]
[4] Jing et al. KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19. Science, 2022. [PubMed]
[5] Shiakolas et al. KIR+CD8+ Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking. Nature Biotechnologies, 2022.[PubMed]
[6] Wu et al. Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level. Cancer Discovery, 2022.[PubMed]
[7] Shomroni et al. A novel single‑cell RNA‑sequencing approach and its applicability connecting genotype to phenotype in ageing disease. Scientific Reports, 2022.[PubMed]
[8] Sole-Boldo et al. Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming. Nature, 2022.[PubMed]
[9] Baek et al. Single-cell transcriptome analysis reveals three sequential phases of gene expression during zebrafish sensory hair cell regeneration. Developmental Cell, 2022.[PubMed]
[10] Satoh et al. A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos. Developmental Biology, 2022.[PubMed]
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