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1.
J Transl Med ; 22(1): 441, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38730481

RESUMO

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.


Assuntos
Morte Celular , Células Gigantes , Interfase , Microtúbulos , Poliploidia , Humanos , Interfase/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Linhagem Celular Tumoral , Morte Celular/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/genética , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
2.
bioRxiv ; 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38645018

RESUMO

Over-activation of the epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR-targeted therapies have led to minimal clinical response. While delivery of EGFR inhibitors (EGFRis) to the brain constitutes a major challenge, how additional drug-specific features alter efficacy remains poorly understood. We apply highly multiplex single-cell chemical genomics to define the molecular response of glioblastoma to EGFRis. Using a deep generative framework, we identify shared and drug-specific transcriptional programs that group EGFRis into distinct molecular classes. We identify programs that differ by the chemical properties of EGFRis, including induction of adaptive transcription and modulation of immunogenic gene expression. Finally, we demonstrate that pro-immunogenic expression changes associated with a subset of tyrphostin family EGFRis increase the ability of T-cells to target glioblastoma cells.

3.
Nat Biotechnol ; 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514799

RESUMO

Spatially resolved gene expression profiling provides insight into tissue organization and cell-cell crosstalk; however, sequencing-based spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for rigorous interpretation of cell states, mostly do not use associated histology images and are not capable of inferring shared neighborhoods across multiple tissues. Here we present Starfysh, a computational toolbox using a deep generative model that incorporates archetypal analysis and any known cell type markers to characterize known or new tissue-specific cell states without a single-cell reference. Starfysh improves the characterization of spatial dynamics in complex tissues using histology images and enables the comparison of niches as spatial hubs across tissues. Integrative analysis of primary estrogen receptor (ER)-positive breast cancer, triple-negative breast cancer (TNBC) and metaplastic breast cancer (MBC) tissues led to the identification of spatial hubs with patient- and disease-specific cell type compositions and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC.

4.
Cell Genom ; 4(2): 100487, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38278156

RESUMO

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.


Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Transdução de Sinais , Receptores Proteína Tirosina Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Genômica , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/uso terapêutico
5.
BMC Genomics ; 24(1): 737, 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38049719

RESUMO

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.


Assuntos
Cromatina , DNA , Cromatina/genética , DNA/genética , Sequenciamento de Cromatina por Imunoprecipitação , Análise de Sequência de DNA/métodos , Epigenômica/métodos
6.
bioRxiv ; 2023 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-37693393

RESUMO

Microtubule targeting agents ( MTAs ) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells can escape death in mitosis and exit mitosis, and become malignant polyploid giant cancer cells ( PGCC ). Considering the low number of malignant cells undergoing mitosis in tumor tissue, killing these cells in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule assembly, preferentially kills cancer cells in interphase as opposed to mitosis, and avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces an integrated stress response and promotes mitochondria fission accompanied by a reduction in energy metabolism. This cell response may underly death in interphase and avoid the development of PGCC.

7.
bioRxiv ; 2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37398090

RESUMO

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

8.
Mol Syst Biol ; 19(6): e11517, 2023 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-37154091

RESUMO

Recent advances in multiplexed single-cell transcriptomics experiments facilitate the high-throughput study of drug and genetic perturbations. However, an exhaustive exploration of the combinatorial perturbation space is experimentally unfeasible. Therefore, computational methods are needed to predict, interpret, and prioritize perturbations. Here, we present the compositional perturbation autoencoder (CPA), which combines the interpretability of linear models with the flexibility of deep-learning approaches for single-cell response modeling. CPA learns to in silico predict transcriptional perturbation response at the single-cell level for unseen dosages, cell types, time points, and species. Using newly generated single-cell drug combination data, we validate that CPA can predict unseen drug combinations while outperforming baseline models. Additionally, the architecture's modularity enables incorporating the chemical representation of the drugs, allowing the prediction of cellular response to completely unseen drugs. Furthermore, CPA is also applicable to genetic combinatorial screens. We demonstrate this by imputing in silico 5,329 missing combinations (97.6% of all possibilities) in a single-cell Perturb-seq experiment with diverse genetic interactions. We envision CPA will facilitate efficient experimental design and hypothesis generation by enabling in silico response prediction at the single-cell level and thus accelerate therapeutic applications using single-cell technologies.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Análise da Expressão Gênica de Célula Única
9.
bioRxiv ; 2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36945538

RESUMO

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

11.
Nat Commun ; 13(1): 2666, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35562344

RESUMO

Single-cell RNA sequencing (scRNA-seq) offers a high-resolution molecular view into complex tissues, but suffers from high levels of technical noise which frustrates efforts to compare the gene expression programs of different cell types. "Spike-in" RNA standards help control for technical variation in scRNA-seq, but using them with recently developed, ultra-scalable scRNA-seq methods based on combinatorial indexing is not feasible. Here, we describe a simple and cost-effective method for normalizing transcript counts and subtracting technical variability that improves differential expression analysis in scRNA-seq. The method affixes a ladder of synthetic single-stranded DNA oligos to each cell that appears in its RNA-seq library. With improved normalization we explore chemical perturbations with broad or highly specific effects on gene regulation, including RNA pol II elongation, histone deacetylation, and activation of the glucocorticoid receptor. Our methods reveal that inhibiting histone deacetylation prevents cells from executing their canonical program of changes following glucocorticoid stimulation.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Perfilação da Expressão Gênica/métodos , Histonas , RNA-Seq , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
12.
Nat Biomed Eng ; 6(4): 463-475, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35478224

RESUMO

The functions of cilia-antenna-like organelles associated with a spectrum of disease states-are poorly understood, particularly in human cells. Here we show that human pluripotent stem cells (hPSCs) edited via CRISPR to knock out the kinesin-2 subunits KIF3A or KIF3B can be used to model ciliopathy phenotypes and to reveal ciliary functions at the tissue scale. KIF3A-/- and KIF3B-/- hPSCs lacked cilia, yet remained robustly self-renewing and pluripotent. Tissues and organoids derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease (PKD) and other features of the ciliopathy spectrum. We also show that human cilia mediate a critical switch in hedgehog signalling during organoid differentiation, and that they constitutively release extracellular vesicles containing signalling molecules associated with ciliopathy phenotypes. The capacity of KIF3A-/- and KIF3B-/- hPSCs to reveal endogenous mechanisms underlying complex ciliary phenotypes may facilitate the discovery of candidate therapeutics.


Assuntos
Ciliopatias , Células-Tronco Pluripotentes , Cílios , Ciliopatias/genética , Proteínas Hedgehog/genética , Humanos , Cinesinas/genética , Fenótipo
13.
Mol Syst Biol ; 17(6): e9522, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34101353

RESUMO

Single-cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA-seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent-like state in neuroepithelial-derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non-dividing neural progenitors. Putative glioblastoma stem-like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down-regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent-like state found in neuroepithelial-derived cells and gliomas.


Assuntos
Glioblastoma , Células-Tronco Neurais , Animais , Ciclo Celular/genética , Divisão Celular , Humanos , Neurogênese/genética
14.
Curr Opin Plant Biol ; 54: 114-121, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32388018

RESUMO

Single-cell genomic approaches have the potential to revolutionize the study of plant systems. Here, we highlight newly developed techniques to analyze transcriptomes at single-cell resolution. We focus on the rigorous standards necessary to generate and compare these data sets introducing analysis methods that can be applied to interpret their results. Lastly, we discuss the inherent limitations of single-cell studies and address future directions for plant single-cell genomics.


Assuntos
Genômica , Plantas/genética , Células Vegetais , Transcriptoma
15.
Cell Syst ; 10(3): 265-274.e11, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32135093

RESUMO

Here, we present Scribe (https://github.com/aristoteleo/Scribe-py), a toolkit for detecting and visualizing causal regulatory interactions between genes and explore the potential for single-cell experiments to power network reconstruction. Scribe employs restricted directed information to determine causality by estimating the strength of information transferred from a potential regulator to its downstream target. We apply Scribe and other leading approaches for causal network reconstruction to several types of single-cell measurements and show that there is a dramatic drop in performance for "pseudotime"-ordered single-cell data compared with true time-series data. We demonstrate that performing causal inference requires temporal coupling between measurements. We show that methods such as "RNA velocity" restore some degree of coupling through an analysis of chromaffin cell fate commitment. These analyses highlight a shortcoming in experimental and computational methods for analyzing gene regulation at single-cell resolution and suggest ways of overcoming it.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Algoritmos , Animais , Diferenciação Celular/genética , Bases de Dados Genéticas , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/fisiologia , Humanos , RNA/genética , Análise de Célula Única/métodos , Software
16.
Science ; 367(6473): 45-51, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31806696

RESUMO

High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.


Assuntos
Ensaios de Triagem em Larga Escala , Neoplasias/metabolismo , RNA-Seq/métodos , Análise de Célula Única/métodos , Transcriptoma/efeitos dos fármacos , Células A549 , Acetatos/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Genômica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células K562 , Células MCF-7 , Neoplasias/genética
17.
Nat Genet ; 51(9): 1389-1398, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477929

RESUMO

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-ß. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.


Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , Testes Genéticos , Glândulas Mamárias Humanas/metabolismo , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
18.
Elife ; 82019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31140974

RESUMO

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Pigmentos Biológicos/metabolismo , Peixe-Zebra
20.
Plant Cell ; 31(5): 993-1011, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30923229

RESUMO

Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis (Arabidopsis thaliana) root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Transcriptoma , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Resposta ao Choque Térmico , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Análise de Sequência de RNA , Análise de Célula Única , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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