Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has ...suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later
. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Studies of acute myeloid leukemia rely on DNA sequencing and immunophenotyping by flow cytometry as primary tools for disease characterization. However, leukemia tumor heterogeneity complicates ...integration of DNA variants and immunophenotypes from separate measurements. Here we introduce DAb-seq, a technology for simultaneous capture of DNA genotype and cell surface phenotype from single cells at high throughput, enabling direct profiling of proteogenomic states in tens of thousands of cells. To demonstrate the approach, we analyze the disease of three patients with leukemia over multiple treatment timepoints and disease recurrences. We observe complex genotype-phenotype dynamics that illustrate the subtlety of the disease process and the degree of incongruity between blast cell genotype and phenotype in different clinical scenarios. Our results highlight the importance of combined single-cell DNA and protein measurements to fully characterize the heterogeneity of leukemia.
Meningiomas are the most common primary intracranial tumors, but the molecular drivers of meningioma tumorigenesis are poorly understood. We hypothesized that investigating intratumor heterogeneity ...in meningiomas would elucidate biologic drivers and reveal new targets for molecular therapy. To test this hypothesis, here we perform multiplatform molecular profiling of 86 spatially-distinct samples from 13 human meningiomas. Our data reveal that regional alterations in chromosome structure underlie clonal transcriptomic, epigenomic, and histopathologic signatures in meningioma. Stereotactic co-registration of sample coordinates to preoperative magnetic resonance images further suggest that high apparent diffusion coefficient (ADC) distinguishes meningioma regions with proliferating cells enriched for developmental gene expression programs. To understand the function of these genes in meningioma, we develop a human cerebral organoid model of meningioma and validate the high ADC marker genes CDH2 and PTPRZ1 as potential targets for meningioma therapy using live imaging, single cell RNA sequencing, CRISPR interference, and pharmacology.
The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 ...deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.
Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. ...However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.
Ependymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples ...from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma.
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•Epigenomic, transcriptomic, and exome profiles and phylogenies within an ependymoma•Spatially distinct clusters reflect neuronal development gene expression programs•Quantitative magnetic resonance imaging characteristics identify stem-like regions•Mutant histone methyltransferase SETD2 enhances proliferation in ependymoma cells
Tumor heterogeneity poses a barrier to cancer treatment. Liu et al. investigate radiographically distinct regions of an ependymoma tumor using transcriptomic, genetic, and epigenomic profiling and discover axes of gene expression programs that recapitulate normal brain development in addition to phylogenies that shed light on the tumorigenesis of ependymoma.
The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell ...genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.
Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it ...remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.
Injectable colloids that self‐assemble into 3D networks are promising materials for applications in regenerative engineering, as they create open systems for cellular infiltration, interaction, and ...activation. However, most injectable colloids have spherical morphologies, which lack the high material‐biology contact areas afforded by higher aspect ratio materials. To address this need, injectable high aspect ratio particles (HARPs) are developed that form 3D networks to enhance scaffold assembly dynamics and cellular interactions. HARPs are functionalized for tunable surface charge through layer‐by‐layer electrostatic assembly. Positively charged chitosan‐HARPs have improved particle suspension dynamics when compared to spherical particles or negatively charged HARPs. Chit‐HARPs are used to improve the suspension dynamics and viability of MIN6 cells in 3D networks. When combined with negatively charged gelatin microsphere (GelMS) porogens, chit‐HARPs reduce GelMS sedimentation and increase overall network suspension, due to a combination of HARP network formation and electrostatic interactions. Lastly, HARPs are functionalized with fibroblast growth factor 2 (FGF2) to highlight their use for growth factor delivery. FGF2‐HARPs increase fibroblast proliferation through a combination of 3D scaffold assembly and growth factor delivery. Taken together, these studies demonstrate the development and diverse uses of high aspect ratio particles as tunable injectable scaffolds for applications in regenerative engineering.
High aspect ratio particles (HARPs) are fabricated from polycaprolactone and functionalized via layer‐by‐layer assembly with chitosan and heparin to develop injectable colloidal scaffolds. HARPs form entangled networks with improved suspension dynamics compared to spherical particles. HARPs can be used to improve cellular dynamics and viability, facilitate mixed particle co‐assemblies, and initiate growth factor delivery for regenerative engineering applications.
Cellular heterogeneity is inherent to many biological systems, across both normal and disease states. For example, diverse ensembles of microbes in the natural environment fulfill distinct roles ...related to nutrient metabolism and gas fixation. In human cancers, genetic and phenotypic heterogeneity is observed among cells originating from a common oncogenic clone. Understanding biological heterogeneity, whether for metabolic engineering applications or the design of cancer therapeutics, begins at the fundamental unit of the organism: a single cell. Droplet microfluidics enables analyses of single cells at a biologically-relevant scale through rapid compartmentalization and manipulation of millions of parallel reactions. In this thesis, I describe the development and application of single-cell genomics platforms leveraging droplet microfluidics to interrogate many individual genomes. These technologies enable single-cell metagenomics and multiomic analysis of single cancer cells, providing new insights into the extent of cellular heterogeneity and its implications across biology.
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