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We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson's disease.
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Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.
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Dermatopatias , Transcriptoma , Humanos , Animais , Camundongos , Pele , Queratinócitos/metabolismo , Epiderme/patologia , Dermatopatias/patologia , Comunicação CelularRESUMO
The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.
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Embrião de Mamíferos , Perfilação da Expressão Gênica , Humanos , Diferenciação Celular/genética , Pulmão , Células-TroncoRESUMO
Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.
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RNA , Transcriptoma , Criança , Masculino , Humanos , Animais , Camundongos , Transcriptoma/genética , RNA Mensageiro , Benchmarking , BioensaioRESUMO
Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
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Células Clonais , Variações do Número de Cópias de DNA , Instabilidade Genômica , Neoplasias , Análise Espacial , Células Clonais/metabolismo , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Detecção Precoce de Câncer , Genoma Humano , Instabilidade Genômica/genética , Genômica , Humanos , Masculino , Modelos Biológicos , Neoplasias/genética , Neoplasias/patologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transcriptoma/genéticaRESUMO
Current methods for spatial transcriptomics are limited by low spatial resolution. Here we introduce a method that integrates spatial gene expression data with histological image data from the same tissue section to infer higher-resolution expression maps. Using a deep generative model, our method characterizes the transcriptome of micrometer-scale anatomical features and can predict spatial gene expression from histology images alone.
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Transcriptoma , Transcriptoma/genéticaRESUMO
Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.
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Senescência Celular , Inversão Cromossômica , Cromossomos/ultraestrutura , Transição Epitelial-Mesenquimal , Neoplasias/genética , Oncogenes , Recombinação Genética , Animais , Brônquios/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular , Transformação Celular Neoplásica , Ritmo Circadiano , Biologia Computacional , Células Epiteliais/metabolismo , Citometria de Fluxo , Genômica , Humanos , Cariotipagem , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Fenótipo , Ligação Proteica , Domínios Proteicos , Fenótipo Secretor Associado à SenescênciaRESUMO
Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3' end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.
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Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.
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DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inclusão em Parafina/métodos , Análise de Sequência de DNA/métodos , Células A549 , Linhagem Celular Tumoral , DNA/isolamento & purificação , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Células HeLa , Humanos , Células MCF-7 , Reprodutibilidade dos TestesRESUMO
DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.
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Sondas de DNA/genética , Bases de Dados de Ácidos Nucleicos , Genoma Humano/genética , Hibridização in Situ Fluorescente/métodos , Células A549 , Mapeamento Cromossômico/métodos , Cromossomos Humanos/genética , Fibroblastos , Células-Tronco Embrionárias Humanas , Humanos , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Projetos de PesquisaRESUMO
Functional validation of candidate genes involved in adaptation and speciation remains challenging. Here, we exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification, we quantified cell-type-specific gene expression in the histological context of growing feather follicles. Expression of Tyrosinase Related Protein 1 (TYRP1), Solute Carrier Family 45 member 2 (SLC45A2) and Hematopoietic Prostaglandin D Synthase (HPGDS) was melanocyte-limited and significantly reduced in follicles from hooded crow, explaining the substantially lower eumelanin content in grey versus black feathers. The central upstream Melanocyte Inducing Transcription Factor (MITF) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations.
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Corvos/fisiologia , Plumas/fisiologia , Regulação da Expressão Gênica , Especiação Genética , Pigmentação/genética , Pigmentos Biológicos/genética , RNA Mensageiro/genética , Animais , Cor , Corvos/genética , Plumas/crescimento & desenvolvimento , Melanócitos/metabolismo , Pigmentos Biológicos/biossínteseRESUMO
BACKGROUND: Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs). RESULTS: We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers. CONCLUSIONS: We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors.
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Quebras de DNA de Cadeia Dupla , Variação Estrutural do Genoma , Modelos Genéticos , Neoplasias/genética , Humanos , Células K562 , Células MCF-7 , Análise de RegressãoRESUMO
Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 × ) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of ~90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.
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DNA double-strand breaks (DSBs) are major DNA lesions that are constantly formed during physiological processes such as DNA replication, transcription, and recombination, or as a result of exogenous agents such as ionizing radiation, radiomimetic drugs, and genome editing nucleases. Unrepaired DSBs threaten genomic stability by leading to the formation of potentially oncogenic rearrangements such as translocations. In past few years, several methods based on next-generation sequencing (NGS) have been developed to study the genome-wide distribution of DSBs or their conversion to translocation events. We developed Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS), which was the first method for direct labeling of DSBs in situ followed by their genome-wide mapping at nucleotide resolution (Crosetto et al., Nat Methods 10:361-365, 2013). Recently, we have further expanded the quantitative nature, applicability, and scalability of BLESS by developing Breaks Labeling In Situ and Sequencing (BLISS) (Yan et al., Nat Commun 8:15058, 2017). Here, we first present an overview of existing methods for genome-wide localization of DSBs, and then focus on the BLESS and BLISS methods, discussing different assay design options depending on the sample type and application.
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Quebras de DNA de Cadeia Dupla , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Edição de Genes , Biblioteca Gênica , Estudo de Associação Genômica Ampla/métodos , Instabilidade Genômica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da PolimeraseRESUMO
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.