RESUMO
Traditionally, flow cytometers acquired data using the same number of detectors as fluorochromes being measured in the experiment. More recently, spectral flow cytometers utilize a larger number of detectors than fluorochromes. This seemingly small difference opens the door to a wide variety of mathematical tools for the calculation of the true fluorochrome abundances from the raw detector values as compared with traditional compensation. This review will provide a brief overview of the mathematics and theory underlying traditional compensation and unmixing focusing on the differences between them and the additional information provided by unmixing approaches.
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Corantes Fluorescentes , Citometria de FluxoRESUMO
Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data-acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least-square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative-error minimization and variance-stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity-link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8-channel, 2-fluorochrome data and real 32-channel, 6-fluorochrome data. The quality of unmixing is assessed by computing absolute and relative errors, as well as by calculating the symmetrized Kullback-Leibler divergence between known and approximated populations. These results are applicable to any flow-based system with more detectors than labels where Poisson noise is the dominant contributor to the overall system noise and highlight the fact that explicit incorporation of appropriate noise models is the key to accurately estimating the true label abundance on the cells. © 2013 International Society for Advancement of Cytometry.
Assuntos
Citometria de Fluxo/métodos , Modelos Lineares , Modelos Estatísticos , Células Sanguíneas/citologia , Corantes Fluorescentes , Humanos , Distribuição de PoissonRESUMO
There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications.
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Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Calibragem , Fenômenos Fisiológicos Celulares , Células , Fluorescência , Corantes Fluorescentes , Raios Infravermelhos , Luz , Microesferas , Pontos Quânticos , Análise de Célula ÚnicaRESUMO
Background: Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo-, and radiotherapy. A deeper understanding of the mechanisms invoked by GBM to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. Methods: CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterized EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived ECM to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. Results: GBM cells expressing a p53 mutant (p53R273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. Conclusions: This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.
RESUMO
Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Mamárias Animais/patologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type.The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.
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Processamento Eletrônico de Dados/normas , Citometria de Fluxo/normas , Biologia Computacional , Processamento Eletrônico de Dados/métodos , Citometria de Fluxo/métodos , Software/normasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features transdifferentiation of tissue-resident pancreatic stellate cells (PSC) into activated cancer-associated fibroblasts, a process induced by PDAC cells but of unclear significance for PDAC progression. Here, we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration, and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound-healing mediators to stimulate their own growth and migration. SIGNIFICANCE: Our work highlights an unanticipated role for PSCs in producing the oncogenic LPA signaling lipid and demonstrates how PDAC tumor cells co-opt the release of wound-healing mediators by neighboring PSCs to promote their own proliferation and migration.See related commentary by Biffi and Tuveson, p. 578.This article is highlighted in the In This Issue feature, p. 565.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Lisofosfatidilcolinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Células Estreladas do Pâncreas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Células Estromais/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Transdução de Sinais , Células Estromais/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Flow cytometry data analysis routinely includes the use of one- or two-parameter histograms to visualize the data. These histograms have traditionally been plotted with either a linear or logarithmic scale. However, the recent trend of performing the logarithmic conversion in software has made apparent some limitations of the traditional visual presentation of logarithmic data. This review discusses the mathematics of presenting data on a histogram and emphasizes the difference between scaling and binning. The review introduces the concept of an effective resolution to describe how the bin width changes in a variable bin-width histogram. The change in effective resolution is used to explain the commonly observed valley and picket fencing artifacts. These result from the effective resolution of the display histogram being too high for the data being presented. Recently, several different binning transformations have been described that are becoming more popular because they allow one to view a large dynamic range of data on a single plot, while allowing the display of negative data values. While each of the transforms is based upon different equations, they all exhibit very similar properties. All of the transforms bin the data logarithmically at high channel values and linearly at low channel values. The linear scaling of the lower channels serves to limit the effective resolution of the histogram, thus minimizing the valley and picket fencing artifacts. The newer transformations are not without their own limitations and recommendations for the appropriate manner of presenting flow cytometry data using these newer transformations are discussed.
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Citometria de Fluxo/métodos , Software , ArtefatosRESUMO
High Content Analysis instrumentation has undergone tremendous hardware advances in recent years. It is now possible to obtain images of hundreds of thousands to millions of individual objects, across multiple wells, channels, and plates, in a reasonable amount of time. In addition, it is possible to extract dozens, or hundreds, of features per object using commonly available software tools. Analyzing this data provides new challenges to the scientists. The magnitude of these numbers is reminiscent of flow cytometer, where practitioners have long been taking what effectively amounted to very low resolution, multi-parametric measurements from individual cells for many decades. Flow cytometrists have developed a wide range of tools to effectively analyze and interpret these types of data. This chapter will review the techniques used in flow cytometry and show how they can easily and effectively be applied to High Content Analysis.
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Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Biomarcadores , Interpretação Estatística de Dados , Citometria de Fluxo/métodos , Humanos , Microscopia , Análise de Célula Única/métodos , SoftwareRESUMO
Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53's invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53's ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth.
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Exossomos/metabolismo , Sialoglicoproteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Exossomos/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Microscopia de Força Atômica , Mutação/genética , Sialoglicoproteínas/genética , Sialomucinas/genética , Sialomucinas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness.
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Neoplasias da Mama/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias Mamárias Animais/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Espaço Extracelular/metabolismo , Feminino , Homeostase , Humanos , Neoplasias Mamárias Animais/patologia , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Invasividade Neoplásica , Receptores de Glutamato Metabotrópico/metabolismo , Regulação para Cima , Proteínas rab27 de Ligação ao GTP/metabolismoRESUMO
Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca2+ release by measuring Ca2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca2+ release elicited by voltage pulses was reduced by approximately 67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca2+ may be altered in the mdx mouse.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular de Duchenne/fisiopatologia , Condução Nervosa/fisiologia , Retículo Sarcoplasmático/fisiologia , Potenciais de Ação/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdxRESUMO
We developed a three-dimensional cylindrical diffusion-reaction model of a single amphibian myofibril in which Ca(2+) release occurred only at the Z-line. The model incorporated diffusion of Ca(2+), Mg(2+), and all relevant buffer species, as well as the kinetic binding reactions between the buffers and appropriate ions. Model data was blurred according to a Gaussian approximation of the point spread function of the microscope and directly compared with experimental data obtained using the confocal spot methodology. The flux parameters were adjusted until the simulated Z-line transient matched the experimental one. This model could not simultaneously predict key parameters of the experimental M- and Z-line transients, even when model parameters were adjusted to unreasonably extreme values. Even though the model was accurate in predicting the Z-line transient under conditions of high [EGTA], it predicted a significantly narrower Ca(2+) domain than observed experimentally. We modified the model to incorporate a broader band of release centered at the Z-line. This extended release model was superior both in simultaneously predicting critical features of the Z- and M-line transients as well as the domain profile under conditions of high [EGTA]. We conclude that a model of release occurring exclusively at the Z-line cannot explain our experimental data and suggest that Ca(2+) may be released from a broader region of the sarcoplasmic reticulum than just the T-tubule-sarcoplasmic reticulum junction.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Magnésio/metabolismo , Modelos Biológicos , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Miofibrilas/fisiologia , Anfíbios , Animais , Transporte Biológico/fisiologia , Simulação por Computador , Difusão , Rana catesbeiana , Retículo Sarcoplasmático/fisiologia , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
The spatiotemporal properties of the Ca2+ release process in skeletal muscle fibres were determined using a confocal spot detection system. The low-affinity, fluorescent Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) was used to record localized, action potential-induced fluorescence signals from consecutive locations separated by 200 nm within a single sarcomere. Three-dimensional reconstructions of the Ca2+ transients illustrated the existence of fluorescence domains around Ca2+ release sites, which are centred at the T-tubules. By constructing isochronal plots, it was estimated that the earliest detectable full width at half-maximum (FWHM) of the Ca2+ domains was 0.77+/-0.08 microm and increased rapidly with time to 1.4+/-0.04 microm at peak (17-18 degrees C). A delay of 0.64+/-0.1 ms was observed between the onset of the fluorescence transients at the Z- and M-lines. Deconvolution of fluorescence transients gave estimates of approximately 9 and 2 microM for the peak [Ca2+] changes at the Z and M-lines, respectively. Our results are compatible with the possibility that action potential stimulation elicits Ca2+ release from a region of the sarcoplasmic reticulum (SR) broader than the T-SR junction.
Assuntos
Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiologia , Potenciais de Ação/fisiologia , Animais , Ácidos Carboxílicos , Quelantes , Eletrofisiologia , Corantes Fluorescentes , Músculo Esquelético/citologia , Rana catesbeiana , Retículo Sarcoplasmático/metabolismoRESUMO
The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca(2+) release is altered in mdx fibres. Action potential-evoked Ca(2+)-dependent fluorescence transients were recorded, using both low and high affinity Ca(2+) indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N-benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca(2+) transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca(2+) transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca(2+) release flux is responsible for the decrease in the peak of the Ca(2+) transient in mdx fibres. Since the myoplasmic Ca(2+) concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.