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A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B , Translocación Genética , Humanos , Edición Génica , Proteómica , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas de Unión al ADN/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.
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Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Dasatinib/farmacología , Dasatinib/uso terapéutico , Receptores de Glucocorticoides/genética , Apoptosis , Proteómica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Recurrencia , Resistencia a Antineoplásicos/genética , Línea Celular TumoralRESUMEN
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.
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Algoritmos , Conjuntos de Datos como Asunto , Citometría de Flujo/métodos , Melanoma/tratamiento farmacológico , Biología Computacional/métodos , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/patología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/patología , Análisis de la Célula Individual , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patologíaRESUMEN
Mass cytometry is now a well-established method that enables the measurement of 40-50 markers (generally proteins but transcripts are also possible) in single cells. Analytes are detected via antibodies tagged with heavy metal and detected by using a time-of-flight mass spectrometer. Over the past decade, mass cytometry has proven to be a valuable method for immunophenotyping hematopoietic cells with remarkable precision in both healthy and malignant scenarios. This chapter explains in detail how to profile hematopoietic cells by using this high-dimensional multiplexed approach.
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Antígenos de Diferenciación/metabolismo , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , InmunofenotipificaciónRESUMEN
We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.
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Linfocitos B/inmunología , Susceptibilidad a Enfermedades , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/diagnóstico , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/etiología , Linfocitos B/metabolismo , Biomarcadores , Biopsia , Preescolar , Citocinas/metabolismo , Susceptibilidad a Enfermedades/inmunología , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Lactante , Recuento de Linfocitos , Masculino , Fenotipo , Piel/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Secuenciación del Exoma , Inactivación del Cromosoma XRESUMEN
Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.
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Anticuerpos/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/análisis , ARN/análisis , Análisis de la Célula Individual/métodos , Animales , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Leucemia Mieloide Aguda/genética , Células Madre/metabolismo , Translocación Genética/genética , Animales , Humanos , RatonesRESUMEN
Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the CRLF2 gene (hiCRLF2) have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.
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In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.
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Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B/clasificación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Análisis de la Célula Individual , Adulto , Linfocitos B/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Recurrencia , Medición de Riesgo , Transducción de Señal , Análisis de Supervivencia , Serina-Treonina Quinasas TOR/metabolismo , Adulto JovenRESUMEN
Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.
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Linaje de la Célula , Separación Celular/métodos , Citometría de Flujo/métodos , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Regeneración , Análisis de la Célula Individual/métodos , Células Madre/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , Venenos Elapídicos/toxicidad , Proteína-1 Reguladora de Fusión/metabolismo , Genes Reporteros , Genotipo , Ensayos Analíticos de Alto Rendimiento , Receptores de Hialuranos/metabolismo , Integrina beta4/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Proteína MioD/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/patología , Factor de Transcripción PAX7/deficiencia , Factor de Transcripción PAX7/genética , Fenotipo , Regeneración/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/patología , Tetraspanina 29/metabolismo , Factores de TiempoRESUMEN
The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.
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Anticuerpos Monoclonales/inmunología , Neoplasias de la Mama/patología , Proteínas de la Cápside/inmunología , Cápside/química , Sistemas de Liberación de Medicamentos/métodos , Receptores de Superficie Celular/inmunología , Virión/inmunología , Anticuerpos Monoclonales/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Cápside/inmunología , Proteínas de la Cápside/metabolismo , Femenino , Citometría de Flujo , Fluorescencia , Humanos , Inmunoconjugados/metabolismo , Microscopía Confocal , Receptores de Superficie Celular/metabolismoRESUMEN
Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.
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Biología Computacional/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatología , Análisis de la Célula Individual/métodos , Médula Ósea/patología , Niño , Estudios de Cohortes , Heterogeneidad Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , TranscriptomaRESUMEN
Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.
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Citometría de Flujo/métodos , Elementos de la Serie de los Lantanoides , Leucocitos Mononucleares/citología , Espectrometría de Masas/métodos , Microesferas , Poliestirenos , Algoritmos , Citometría de Flujo/instrumentación , Humanos , Espectrometría de Masas/instrumentación , Ensayo de Materiales/métodos , Control de Calidad , Valores de Referencia , Programas InformáticosRESUMEN
Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.