scholarly journals Comprehensive Integration of Single-Cell Transcriptional Profiling Reveals the Heterogeneities of Non-cardiomyocytes in Healthy and Ischemic Hearts

2020 ◽  
Vol 7 ◽  
Author(s):  
Lingfang Zhuang ◽  
Lin Lu ◽  
Ruiyan Zhang ◽  
Kang Chen ◽  
Xiaoxiang Yan
Keyword(s):  
Myocardial Infarction ◽  
Single Cell ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Developmental Trajectories ◽  
Transcriptional Profiling ◽  
Sequencing Data ◽  
Signature Genes

Advances in single-cell RNA sequencing (scRNA-seq) technology have recently shed light on the molecular mechanisms of the spatial and temporal changes of thousands of cells simultaneously under homeostatic and ischemic conditions. The aim of this study is to investigate whether it is possible to integrate multiple similar scRNA-seq datasets for a more comprehensive understanding of diseases. In this study, we integrated three representative scRNA-seq datasets of 27,349 non-cardiomyocytes isolated at 3 and 7 days after myocardial infarction or sham surgery. In total, seven lineages, including macrophages, fibroblasts, endothelia, and lymphocytes, were identified in this analysis with distinct dynamic and functional properties in healthy and nonhealthy hearts. Myofibroblasts and endothelia were recognized as the central hubs of cellular communication via ligand-receptor interactions. Additionally, we showed that macrophages from different origins exhibited divergent transcriptional signatures, pathways, developmental trajectories, and transcriptional regulons. It was found that myofibroblasts predominantly expand at 7 days after myocardial infarction with pro-reparative characteristics. We identified signature genes of myofibroblasts, such as Postn, Cthrc1, and Ddah1, among which Ddah1 was exclusively expressed on activated fibroblasts and exhibited concordant upregulation in bulk RNA sequencing data and in vivo and in vitro experiments. Collectively, this compendium of scRNA-seq data provides a valuable entry point for understanding the transcriptional and dynamic changes of non-cardiomyocytes in healthy and nonhealthy hearts by integrating multiple datasets.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

EPCO-18. A MESENCHYMAL CELL POPULATION MEDIATES RESISTANCE TO AURORA KINASE INHIBITORS IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi5-vi5
Author(s):  
Robert Suter ◽  
Vasileios Stathias ◽  
Anna Jermakowicz ◽  
Hari Pradhyumnan ◽  
Maurizio Affer ◽  
...  
Keyword(s):  
Single Cell ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Patient Survival ◽  
Aurora Kinase ◽  
Adult Brain ◽  
Sequencing Data ◽  
Kinase Inhibition

Abstract Glioblastoma (GBM) remains the most common adult brain cancer, with a dismal average patient survival of less than two years. No new treatments have been approved for GBM since the introduction of the alkylating agent temozolomide in 2005. Even then, temozolomide treatment only increases the average survival of GBM patients by a few months. Thus, novel therapeutic options are direly needed. The aurora kinases A and B are targetable and overexpressed in GBM, and their expression is highly correlated with patient survival outcomes. Our lab has found that small molecule aurora kinase inhibition reduces GBM tumor growth in vitro and in vivo, however, eventually tumors still grow. Computational analysis integrating compound transcriptional response signatures from the LINCS L1000 dataset with the single-cell RNA-sequencing data of patient GBM tumors resected at the University of Miami predicts that aurora inhibition targets a subset of cells present within any GBM tumor. Results of in vivo single-cell perturbation experiments with the aurora kinase inhibitor alisertib coincide with our predictions and reveal a cellular transcriptional phenotype resistant to aurora kinase inhibition, characterized by a mesenchymal expression program. We find that small molecules that are predicted to target different cell populations from alisertib, including this resistant mesenchymal population, synergize with alisertib to kill GBM cells. As a whole, we have identified the cellular population resistant to aurora kinase inhibition and have developed an analytical framework that identifies synergistic small molecule combinations by identifying compounds that target transcriptionally distinct cellular populations within GBM tumors.

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

scholarly journals The novel BET inhibitor UM-002 reduces glioblastoma cell proliferation and invasion

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna M. Jermakowicz ◽  
Matthew J. Rybin ◽  
Robert K. Suter ◽  
Jann N. Sarkaria ◽  
Zane Zeier ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Adult Brain ◽  
Bet Inhibitor ◽  
Bet Inhibitors ◽  
Single Cell Rna Sequencing

AbstractBromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.

scholarly journals DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation

2021 ◽  
pp. jmedgenet-2020-107447
Author(s):  
Lev Prasov ◽  
Brenda L Bohnsack ◽  
Antonette S El Husny ◽  
Lam C Tsoi ◽  
Bin Guan ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Skin Rash ◽  
Open Angle Glaucoma ◽  
Response To Therapy ◽  
Functional Characterisation ◽  
Single Cell Rna Sequencing ◽  
Rna Ligands

BackgroundSingleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2.MethodsFamilies underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed.ResultsWe have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin.ConclusionsThese results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

215 INVESTIGATION OF A PREFERENTIALLY UPREGULATED GENE CLUSTER IN DAY 7 BOVINE EMBRYOS DERIVED FROM RNA SEQUENCING DATA

2013 ◽  
Vol 25 (1) ◽  
pp. 256 ◽  
Author(s):  
A. Al Naib ◽  
S. Mamo ◽  
P. Lonergan
Keyword(s):  
Rna Sequencing ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Sequencing Data ◽  
Cell Stage ◽  
Uterine Endometrium ◽  
Embryonic Origin

Successful establishment and maintenance of pregnancy requires optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. A recent RNA sequencing study from our group (Mamo et al. 2011 Biol. Reprod. 85, 1143–1151) described the global transcriptome profile of the bovine conceptus at 5 key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using RNA sequencing techniques. One cluster of genes (n = 1680 transcripts) was preferentially upregulated at Day 7 and subsequently downregulated, suggesting that these genes might be markers of blastocyst formation. The objective of this study was to characterise the pattern of expression of these genes before Day 7 (i.e. from the zygote to blastocyst stage). The list of genes was submitted to DAVID (Database for Annotation, Visualisation, and Integrated Discovery) to take advantage of available ontology information contained therein. The expression of 9 genes belonging to ontologies specifically related to embryo developmental (GINS1, TAF8, ESRRB, NCAPG2, SP1, XAB2, CDC2L1, MSX1, and AQP3) plus Na/K ATPase, a gene previously known to be involved in blastocoe formation, was studied by quantitative real-time PCR (QPCR) in 6 replicate pools of 5 embryos produced by maturation, fertilization, and embryo culture in vitro. Stages studies included immature and mature oocyte, zygote, 2- cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, and hatched blastocyst. In addition, in vivo derived Day 13 and Day 16 embryos were included as controls to confirm down-regulation after Day 7. Data were analysed using the GLM procedure of SAS. The QPCR expression data supported the RNA Seq data in that expression of all transcripts was downregulated after the blastocyst stage. Expression before the blastocyst stage was characterised by 1 of 3 broad patterns: (1) the expression was of maternal origin where the expression was very high up to 8-cell stage and decreased subsequently (MSX1), (2) the expression was of embryonic origin being low up to the 8-cell stage and increasing thereafter (TAF8, ESRRB, AQP3, and Na/K ATPase), or (3) static or decreased expression from oocyte to the maternal-zygotic transition followed by increased expression from the 16-cell stage (GINS1, NCAPG2, SP1, XAB2, and CDC2L1). In conclusion, the genes identified in this cluster, despite having different patterns of expression before the blastocyst stage, may represent markers of blastocyst formation in cattle given their downregulation subsequently. Supported by Science Foundation Ireland (07/SRC/B1156).

scholarly journals Robust transcriptional profiling and identification of differentially expressed genes with low input RNA sequencing of adult hippocampal neural stem and progenitor populations

2021 ◽  
Author(s):  
Jiyeon Kim Denninger ◽  
Logan A Walker ◽  
Xi Chen ◽  
Altan M Turkoglu ◽  
Alexander Pan ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Transcriptional Profiling ◽  
Population Level ◽  
Similar Efficacy ◽  
Differentially Expressed ◽  
Mammalian Brain ◽  
Single Cell Sequencing

Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing (RNAseq) of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNAseq can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. The consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains largely unexplored. Here, to clarify the role that low RNA input plays in NSC DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing DEGs derived from genes with higher relative transcript counts compared to all detected genes and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to standard bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work suggests that single cell RNA sequencing may underestimate the diversity of pathologic DEGs but population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as standard bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.

scholarly journals TMIC-59. INVESTIGATING PHYSIOLOGY OF THERAPY RESISTANT, TUMOR-INITIATING GBM CELLS IN HYPOXIC NICHES USING SPATIALLY-SENSITIVE HYPOXIA REPORTER SYSTEM AND SINGLE-CELL RNA SEQUENCING

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi260-vi261
Author(s):  
Anirudh Sattiraju ◽  
Valerie Marallano ◽  
Roland Friedel ◽  
Hongyan Zou
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Reporter System ◽  
T Cell Infiltration ◽  
Single Cell Rna Sequencing ◽  
Stem Cell Lines

Abstract Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.

248 Immunotherapy persister cells uncovered by dynamic single-cell RNA-sequencing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A268-A269
Author(s):  
Kartik Sehgal ◽  
Andrew Portell ◽  
Elena Ivanova ◽  
Patrick Lizotte ◽  
Navin Mahadevan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Interferon Γ ◽  
Rna Seq ◽  
Persister Cells ◽  
Pre Treatment

BackgroundTo understand fundamental mechanisms of immune escape, we leveraged our functional ex vivo platform of murine derived organotypic tumor spheroids (DOTS)1 to determine if drug-tolerant persister cells analogous to oncogene targeted therapies limit efficacy of programmed death (PD)-1 blockade, and to identify therapeutic vulnerabilities to overcome anti-PD-1 (αPD-1) resistance.MethodsMurine syngeneic cancer models with well-characterized response to αPD-1 therapy were chosen: MC38 (sensitive) and CT26 (partially resistant). Bulk and single-cell (sc) RNA-sequencing (RNA-seq) were performed on αPD-1 treated DOTS. In vitro culture studies were conducted with or without cytokines (100 ng/ml) or drugs (500 nM). In vivo studies in mice bearing MC38 or CT26 tumors evaluated the combinatorial strategy with PD-1 blockade. We further evaluated our findings in scRNA-seq of an αPD-1 refractory colorectal cancer (CRC) patient tumor.2ResultsBulk RNA-seq of αPD-1 treated DOTS revealed a mesenchymal resistant phenotype with upregulated TNF-α/NFκB signaling (figure 1). scRNA-seq further identified a discrete sub-population of immunotherapy persister cells (IPCs). These cells expressed a stem-like phenotype including downregulation of E2F targets indicative of quiescence, suppression of interferon-γ response genes, induction of hybrid epithelial-to-mesenchymal state, and active IL-6 signaling (figure 1). Ly6a/stem cell antigen-1 (Sca-1) and Snai1 were found to be differentially upregulated in IPCs resistant to PD-1 blockade (not shown). Sca-1 positivity was confirmed in pre-existing tumor populations in vitro (figure 2). When enriched via sorting, these cells remained more persistently Sca-1+ at 96 hours in culture of CT26 compared to MC38 cells, related to increased autocrine IL-6 production by CT26 Sca-1+ cells. Indeed, IL-6 supplementation was capable of expanding Sca-1+ cells in culture (figure 2). Sca-1+ cells expressing ovalbumin peptide were refractory to OT-1 T cell mediated killing and failed to upregulate MHC class-1 antigen presentation (H-2Kb) in response to IL-6, in contrast to interferon-γ (not shown). Analysis of RNA-seq data further identified Birc2/3 as potential targets limiting TNF-mediated apoptosis of these cells (not shown). Notably, Birc2/3 antagonism depleted Sca-1+ IPCs in vitro and significantly potentiated the impact of PD-1 blockade in vivo in MC38, and less robustly in CT26 (figure 3). Evaluation in a microsatellite-instability high CRC patient identified a pre-existent IPC subpopulation within the αPD-1 refractory pre-treatment tumor, with high SNAI1 expression compared to CRC samples in TCGA (figure 4).Abstract 248 Figure 1Bulk and single-cell (sc) RNA-sequencing (RNA-seq) of MDOTS identifies an anti-PD-1 (αPD-1) resistant subpopulation of persister cells. IgG= isotype controlAbstract 248 Figure 2Pre-existent population of stem cell antigen-1 (Sca-1)+ cells expands in response to interleukin-6 (IL-6), as characterized by flow cytometry evaluation in murine syngeneic cancer models at baseline and after purification by fluorescence-activated cell sorting (FACS). H = hoursAbstract 248 Figure 3Combination of anti-PD-1 therapy with Birc2/3 antagonism increases tumor responses and improves survival. CR = complete responseAbstract 248 Figure 4Single-cell RNA-sequencing (scRNA-seq) of a pre-treatment microsatellite-instability (MSI-H) colorectal cancer (CRC) patient tumor, refractory to anti-PD-1 (αPD-1) therapy, reveals presence of SNAI1-high immunotherapy persister cellsConclusionsHigh-resolution functional ex vivo profiling identified Sca-1+/Snai1high stem-like ‘immunotherapy persister cells‘ and uncovered their anti-apoptotic dependencies targetable with Birc2/3 antagonism to augment αPD-1 efficacy.Ethics ApprovalThis study was approved by the Dana-Farber Animal Care and Use Committee and Novartis Institutional Animal Care and Use Committee. Informed written consent to participate in Dana-Farber/Harvard Cancer Center institutional review board (IRB)-approved research protocols was obtained from the human subject. A copy of the written consent is available for review by the Editor of this journal. The study was conducted per the WMA Declaration of Helsinki and IRB-approved protocols.ReferencesJenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Profiling of PD-1 Blockade using organotypic tumor spheroids. Cancer Discov. 2018;8(2):196–668 215.Gurjao C, Liu D, Hofree M, AlDubayan SH, Wakiro I, Su MJ, et al. intrinsic resistance to immune checkpoint blockade in a mismatch repair-deficient colorectal cancer. Cancer Immunol Res 2019;7(8):1230–6.

scholarly journals Single cell transcriptomic analysis revealed long-lasting adverse effects of prenatal tamoxifen administration on neurogenesis in prenatal and adult brains

2019 ◽  
Author(s):  
Chia-Ming Lee ◽  
Liqiang Zhou ◽  
Jiping Liu ◽  
Jiayu Shi ◽  
Yanan Geng ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Lineage Tracing ◽  
High Rate ◽  
Neural Lineage ◽  
Cortical Neurogenesis

SummaryCreER/LoxP system has enabled precise gene manipulation in distinct cell subpopulations at any specific time point upon tamoxifen (TAM) administration. This system is widely accepted to track neural lineages and study gene functions. We have observed prenatal TAM treatment caused high rate of delayed delivery and mortality of pups. These substances could promote undesired results, leading to data misinterpretation. Here, we report that TAM administration during early stages of cortical neurogenesis promoted precocious neural differentiation, while inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, and cortical patterning in neonatal and postnatal offspring. Mechanistically, single cell RNA sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays showed TAM could exert these drastic effects mainly through dysregulating the expression of Dmrta2 and Wnt8b. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of prenatal tamoxifen administration on corticogenesis, suggesting that tamoxifen-induced CreER/LoxP system may not be suitable for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.SignificantFor the first time, our study revealed the molecular mechanisms underlying tamoxifen activities on cortical development. This study also clearly showed that care must be taken when using tamoxifen-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies.

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