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While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy ("PRINCE", NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response.
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Biomarcadores de Tumor , Carcinoma Ductal Pancreático , ADN Tumoral Circulante , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Femenino , Masculino , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Persona de Mediana Edad , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Pronóstico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mutación , Supervivencia sin Progresión , Metástasis de la NeoplasiaRESUMEN
We analyzed the structure of human long non-coding RNA (lncRNAs) genes to investigate whether the non-coding transcriptome is organized in modular domains, as is the case for protein-coding genes. To this aim, we compared all known human lncRNA exons and identified 340 pairs of exons with high sequence and/or secondary structure similarity but embedded in a dissimilar sequence context. We grouped these pairs in 106 clusters based on their reciprocal similarities. These shared modules are highly conserved between humans and the four great ape species, display evidence of purifying selection and likely arose as a result of recent segmental duplications. Our analysis contributes to the understanding of the mechanisms driving the evolution of the non-coding genome and suggests additional strategies towards deciphering the functional complexity of this class of molecules.
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Cardiovascular diseases (CVDs) represent a major concern for global health, whose mechanistic understanding is complicated by a complex interplay between genetic predisposition and environmental factors. Specifically, heart failure (HF), encompassing dilated cardiomyopathy (DC), ischemic cardiomyopathy (ICM), and hypertrophic cardiomyopathy (HCM), is a topic of substantial interest in basic and clinical research. Here, we used a Partial Correlation Coefficient-based algorithm (PCC) within the context of a meta-analysis framework to construct a Gene Regulatory Network (GRN) that identifies key regulators whose activity is perturbed in Heart Failure. By integrating data from multiple independent studies, our approach unveiled crucial regulatory associations between transcription factors (TFs) and structural genes, emphasizing their pivotal roles in regulating metabolic pathways, such as fatty acid metabolism, oxidative stress response, epithelial-to-mesenchymal transition, and coagulation. In addition to known associations, our analysis also identified novel regulators, including the identification of TFs FPM315 and OVOL2, which are implicated in dilated cardiomyopathies, and TEAD1 and TEAD2 in both dilated and ischemic cardiomyopathies. Moreover, we uncovered alterations in adipogenesis and oxidative phosphorylation pathways in hypertrophic cardiomyopathy and discovered a role for IL2 STAT5 signaling in heart failure. Our findings underscore the importance of TF activity in the initiation and progression of cardiac disease, highlighting their potential as pharmacological targets.
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Enfermedades Cardiovasculares , Redes Reguladoras de Genes , Factores de Transcripción , Humanos , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación de la Expresión Génica , Algoritmos , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismoRESUMEN
Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC ( NCT03214250 ). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.
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Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Albúminas , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Humanos , Nivolumab/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
BACKGROUND: There are no validated biomarkers that can aid clinicians in selecting who would best benefit from anticytotoxic T lymphocyte-associated antigen 4 monotherapy versus combination checkpoint blockade in patients with advanced melanoma who have progressive disease after programmed death 1 (PD-1) blockade. METHODS: We conducted a randomized multicenter phase II trial in patients with advanced melanoma. Patients were randomly assigned to receive either 1 mg/kg of nivolumab plus 3 mg/kg of ipilimumab or 3 mg/kg of ipilimumab every 3 weeks for up to four doses. Patients were stratified by histological subtype and prior response to PD-1 therapy. The primary clinical objective was overall response rate by week 18. Translational biomarker analyses were conducted in patients with blood and tissue samples. RESULTS: Objective responses were seen in 5 of 9 patients in the ipilimumab arm and 2 of 10 patients in the ipilimumab+nivolumab arm; disease control rates (DCRs) (66.7% vs 60.0%) and rates of grade 3-4 adverse events (56% vs 50%) were comparable between arms. In a pooled analysis, patients with clinical benefit (CB), defined as Response Evaluation Criteria in Solid Tumors response or progression-free for 6 months, showed increased circulating CD4+ T cells with higher polyfunctionality and interferon gamma production following treatment. Tumor profiling revealed enrichment of NRAS mutations and activation of transcriptional programs associated with innate and adaptive immunity in patients with CB. CONCLUSIONS: In patients with advanced melanoma that previously progressed on PD-1 blockade, objective responses were seen in both arms, with comparable DCRs. Findings from biomarker analyses provided hypothesis-generating signals for validation in future studies of larger patient cohorts. TRIAL REGISTRATION NUMBER: NCT02731729.
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Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ipilimumab/uso terapéutico , Melanoma/tratamiento farmacológico , Nivolumab/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Presentación de Antígeno , Biomarcadores de Tumor , Femenino , Humanos , Interferón gamma/biosíntesis , Ipilimumab/administración & dosificación , Ipilimumab/efectos adversos , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Nivolumab/efectos adversos , Estudios Prospectivos , Análisis de Secuencia de ARN , Microambiente TumoralRESUMEN
Complex datasets provide opportunities for discoveries beyond their initial scope. Effective and rapid data sharing and management practices are crucial to realize this potential; however, they are harder to implement than post-publication access. Here, we introduce the concept of a "data sharing trust" to maximize the value of large datasets.
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Conducta Cooperativa , Difusión de la Información , Modelos Teóricos , Confianza , Autoria , Humanos , InvestigadoresRESUMEN
BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.
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Adenocarcinoma/tratamiento farmacológico , Albúminas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígenos CD40/antagonistas & inhibidores , Desoxicitidina/análogos & derivados , Nivolumab/administración & dosificación , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/secundario , Anciano , Albúminas/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Antígenos CD40/inmunología , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nivolumab/efectos adversos , Paclitaxel/efectos adversos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Factores de Tiempo , Resultado del Tratamiento , Estados Unidos , GemcitabinaRESUMEN
Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Femenino , Humanos , MasculinoRESUMEN
The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.
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Ensayos Clínicos como Asunto , Inmunofenotipificación/métodos , Inmunoterapia/métodos , Monitorización Inmunológica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Femenino , Enfermedad Injerto contra Huésped/inmunología , Humanos , Inmunofenotipificación/normas , Masculino , Persona de Mediana Edad , Monitorización Inmunológica/normas , Neoplasias/inmunología , Neoplasias/terapia , Estándares de ReferenciaRESUMEN
Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry. We provide detailed instructions for combining this technique with antibody-based detection of surface/internal protein, allowing simultaneous highly multiplexed profiling of RNA and protein expression at single-cell resolution. PLAYR overcomes limitations on multiplexing seen in previous branching DNA-based RNA detection techniques by integration of a transcript-specific oligonucleotide sequence within a rolling-circle amplification (RCA). This unique transcript-associated sequence can then be detected by heavy metal (for mass cytometry)- or fluorophore (for flow cytometry)-conjugated complementary detection oligonucleotides. Included in this protocol is methodology to label oligonucleotides with lanthanide metals for use in mass cytometry. When analyzed by mass cytometry, up to 40 variables (with scope for future expansion) can be measured simultaneously. We used the described protocol to demonstrate intraclonal heterogeneity within primary cells from chronic lymphocytic leukemia patients, but it can be adapted to other primary cells or cell lines in suspension. This robust, reliable and reproducible protocol can be completed in 2-3 d and can be paused at several stages for convenience.
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Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Proteínas/genética , ARN/genética , Animales , Anticuerpos/metabolismo , Antígenos/metabolismo , Linfocitos B/metabolismo , Simulación por Computador , Humanos , Ionomicina/farmacología , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
While meta-analysis has demonstrated increased statistical power and more robust estimations in studies, the application of this commonly accepted methodology to cytometry data has been challenging. Different cytometry studies often involve diverse sets of markers. Moreover, the detected values of the same marker are inconsistent between studies due to different experimental designs and cytometer configurations. As a result, the cell subsets identified by existing auto-gating methods cannot be directly compared across studies. We developed MetaCyto for automated meta-analysis of both flow and mass cytometry (CyTOF) data. By combining clustering methods with a silhouette scanning method, MetaCyto is able to identify commonly labeled cell subsets across studies, thus enabling meta-analysis. Applying MetaCyto across a set of ten heterogeneous cytometry studies totaling 2,926 samples enabled us to identify multiple cell populations exhibiting differences in abundance between demographic groups. Software is released to the public through Bioconductor (http://bioconductor.org/packages/release/bioc/html/MetaCyto.html).
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Citometría de Flujo/métodos , Metaanálisis como Asunto , Programas Informáticos , Adulto , Conjuntos de Datos como Asunto , HumanosRESUMEN
Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging. Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology. Availability and implementation: The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder. Supplementary information: Supplementary data are available at Bioinformatics online.
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Algoritmos , Biomarcadores/análisis , Citometría de Flujo , Programas InformáticosRESUMEN
Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic- and epigenetic-guided mass cytometry approach to define core exhaustion-specific genes and disease-induced changes in Tex cells in HIV and human cancer. Single-cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co-expression patterns. An exhaustion severity metric was developed and integrated with high-dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer.
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Linfocitos T CD8-positivos/inmunología , Epigenómica/métodos , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Infecciones por VIH/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteómica/métodos , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Desmoplastic melanoma is a rare subtype of melanoma characterized by dense fibrous stroma, resistance to chemotherapy and a lack of actionable driver mutations, and is highly associated with ultraviolet light-induced DNA damage. We analysed sixty patients with advanced desmoplastic melanoma who had been treated with antibodies to block programmed cell death 1 (PD-1) or PD-1 ligand (PD-L1). Objective tumour responses were observed in forty-two of the sixty patients (70%; 95% confidence interval 57-81%), including nineteen patients (32%) with a complete response. Whole-exome sequencing revealed a high mutational load and frequent NF1 mutations (fourteen out of seventeen cases) in these tumours. Immunohistochemistry analysis from nineteen desmoplastic melanomas and thirteen non-desmoplastic melanomas revealed a higher percentage of PD-L1-positive cells in the tumour parenchyma in desmoplastic melanomas (P = 0.04); these cells were highly associated with increased CD8 density and PD-L1 expression in the tumour invasive margin. Therefore, patients with advanced desmoplastic melanoma derive substantial clinical benefit from PD-1 or PD-L1 immune checkpoint blockade therapy, even though desmoplastic melanoma is defined by its dense desmoplastic fibrous stroma. The benefit is likely to result from the high mutational burden and a frequent pre-existing adaptive immune response limited by PD-L1 expression.
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Inmunoterapia , Melanoma/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Biopsia , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Puntos de Control del Ciclo Celular , Humanos , Melanoma/genética , Melanoma/metabolismo , Mutación/genética , Neurofibromina 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Estudios RetrospectivosRESUMEN
Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.
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Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Subgrupos de Linfocitos T/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Médula Ósea/inmunología , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Tejido Linfoide/inmunología , Masculino , Melanoma/inmunología , Melanoma/terapia , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Microambiente TumoralRESUMEN
To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating to localization of proteins to and within organelles. We used SLA to detect the nuclear import of transcription factors across cell subsets in complex samples. We further measured intranuclear re-localization of target proteins across the cell cycle and upon DNA damage induction. SLA combines multiple single-cell methods to bring about a new dimension of inquiry and analysis in complex cell populations. © 2017 International Society for Advancement of Cytometry.
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Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestructura , Daño del ADN/genética , Humanos , Transporte de Proteínas/genética , Fracciones Subcelulares/ultraestructuraRESUMEN
Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4+ and CD8+ T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet+CD4+ T cells, CD8+ T cells, B cells, and CD56loCD16+ NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunidad Innata/fisiología , Células Asesinas Naturales/inmunología , Placenta/inmunología , Embarazo/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Femenino , Humanos , Proteínas Gestacionales/inmunología , Factor de Transcripción STAT1/inmunologíaRESUMEN
Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin.
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Neoplasias de la Mama , Diabetes Mellitus Tipo 2 , Humanos , Hipoglucemiantes , Metformina , ProteómicaRESUMEN
To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.
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Citometría de Flujo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis por Matrices de Proteínas/métodos , Proteínas/metabolismo , ARN/metabolismo , Humanos , Células Jurkat , Proteínas/análisis , ARN/análisisRESUMEN
Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.