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1.
Nat Commun ; 9(1): 4550, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382095

RESUMO

Serial tissue sampling has become essential in guiding modern targeted and personalized cancer treatments. An alternative to image guided core biopsies are fine needle aspirates (FNA) that yield cells rather than tissues but are much better tolerated and have lower complication rates. The efficient pathway analysis of such cells in the clinic has been difficult, time consuming and costly. Here we develop an antibody-DNA barcoding approach where harvested cells can be rapidly re-stained through the use of custom designed oligonucleotide-fluorophore conjugates. We show that this approach can be used to interrogate drug-relevant pathways in scant clinical samples. Using the PI3K/PTEN/CDK4/6 pathways in breast cancer as an example, we demonstrate how analysis can be performed in tandem with trial enrollment and can evaluate downstream signaling following therapeutic inhibition. This approach should allow more widespread use of scant single cell material in clinical samples.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Transdução de Sinais , Análise de Célula Única/métodos , Anticorpos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Resultado do Tratamento
2.
Nat Chem Biol ; 13(2): 168-173, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27918558

RESUMO

Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.


Assuntos
Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Análise de Célula Única , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Especificidade por Substrato , Células Tumorais Cultivadas
3.
Sci Rep ; 6: 32985, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27609668

RESUMO

Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations' morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.


Assuntos
Biomarcadores/análise , Monitoramento de Medicamentos/métodos , Processamento de Imagem Assistida por Computador/métodos , Dinâmica Mitocondrial , Neoplasias/patologia , Patologia/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Transplante de Neoplasias
4.
Chem Commun (Camb) ; 52(64): 9953-6, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27439765

RESUMO

Herein we describe the synthesis of several fluorescent analogues of the clinically approved microtubule destabilizing agent vinblastine. The evaluated probes are the most potent described and provides the first example of uptake, distribution and live cell imaging using this well known antimitotic agent.


Assuntos
Corantes Fluorescentes/análise , Vimblastina/análise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Microscopia de Fluorescência/métodos , Estrutura Secundária de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Vimblastina/metabolismo , Vimblastina/farmacologia
5.
Theranostics ; 5(8): 796-804, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26000053

RESUMO

Molecular profiling of central nervous system lymphomas in cerebrospinal fluid (CSF) samples can be challenging due to the paucicellular and limited nature of the samples. Presented herein is a microfluidic platform for complete CSF lymphoid cell analysis, including single cell capture in sub-nanoliter traps, and molecular and chemotherapeutic response profiling via on-chip imaging, all in less than one hour. The system can detect scant lymphoma cells and quantitate their kappa/lambda immunoglobulin light chain restriction patterns. The approach can be further customized for measurement of additional biomarkers, such as those for differential diagnosis of lymphoma subtypes or for prognosis, as well as for imaging exposure to experimental drugs.


Assuntos
Neoplasias do Sistema Nervoso Central/diagnóstico , Líquido Cefalorraquidiano/citologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Linfoma/diagnóstico , Microfluídica/métodos , Neoplasias do Sistema Nervoso Central/patologia , Humanos , Linfoma/patologia
6.
Bioconjug Chem ; 25(11): 2081-5, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25333750

RESUMO

Overexpression of anti-apoptotic proteins such as Bcl-2 is a cellular mechanism to evade apoptosis; consequently, Bcl-2 inhibitors are being developed as anticancer agents. In this work, we have synthesized a fluorescent version of ABT-199 in an effort to visualize a drug surrogate by high resolution imaging. We show that this fluorescent conjugate has comparable Bcl-2 binding efficacy and cell line potency to the parent compound and can be used as an imaging agent in several cancer cell types. We anticipate that this agent will be a valuable tool for studying the single-cell distribution and pharmacokinetics of ABT-199 as well the broader group of BH3-mimetics.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Corantes Fluorescentes/metabolismo , Imagem Molecular/métodos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/metabolismo , Apoptose , Transporte Biológico , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Humanos , Espaço Intracelular/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Sulfonamidas/química , Sulfonamidas/farmacologia
7.
Angew Chem Int Ed Engl ; 53(29): 7531-4, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24915832

RESUMO

We have developed a series of new ultrafluorogenic probes in the blue-green region of the visible-light spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans-cyclooctene(TCO)-tetrazine chemistry platform. By exploiting highly efficient through-bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell-surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.


Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Compostos Heterocíclicos/química , Linhagem Celular
8.
Chem Commun (Camb) ; 50(34): 4504-7, 2014 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-24663433

RESUMO

Here we evaluated a series of Si-derivatized rhodamine (SiR) dyes for their ability to visualize a model drug in live cells. We show that a charge neutral SiR derivative (but not others) can indeed be used to follow the intracellular location of the model therapeutic drug in GFP cells.


Assuntos
Rodaminas/química , Silício/química , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Ftalazinas/química , Piperazinas/química
9.
Intravital ; 3(2): e29917, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-28243510

RESUMO

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

10.
Intravital ; 2(3)2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24349895

RESUMO

Longitudinal analyses of single cell lineages over prolonged periods have been challenging particularly in processes characterized by high cell turn-over such as inflammation, proliferation, or cancer. RGB marking has emerged as an elegant approach for enabling such investigations. However, methods for automated image analysis continue to be lacking. Here, to address this, we created a number of different multicolored poly- and monoclonal cancer cell lines for in vitro and in vivo use. To classify these cells in large scale data sets, we subsequently developed and tested an automated algorithm based on hue selection. Our results showed that this method allows accurate analyses at a fraction of the computational time required by more complex color classification methods. Moreover, the methodology should be broadly applicable to both in vitro and in vivo analyses.

11.
PLoS One ; 8(4): e60988, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23593370

RESUMO

Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level.


Assuntos
Preparações Farmacêuticas/administração & dosagem , Análise de Célula Única , Algoritmos , Linhagem Celular Tumoral , Humanos , Distribuição Tecidual
12.
Ann Biomed Eng ; 40(9): 1903-16, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22527011

RESUMO

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨ(m)), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for "donut" formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.


Assuntos
Metabolismo Energético , Células Endoteliais da Veia Umbilical Humana/fisiologia , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Trifosfato de Adenosina/fisiologia , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Potencial da Membrana Mitocondrial
13.
Free Radic Biol Med ; 52(2): 348-56, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22100972

RESUMO

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to be due to mitochondrial reactive oxygen species (mtROS) production. MtROS have been implicated in mitochondrial fission. We determined whether cultured EC exposure to simulated I/RP causes morphological changes in the mitochondrial network and the mechanisms behind those changes. Because shear stress results in nitric oxide (NO)-mediated endothelial mtROS generation, we simulated I/RP as hypoxia (H) followed by oxygenated flow over the ECs (shear stress of 10dyn/cm(2)). By exposing ECs to shear stress, H, H/reoxygenation (RO), or simulated I/RP and employing MitoTracker staining, we assessed the differential effects of changes in mechanical forces and/or O(2) levels on the mitochondrial network. Static or sheared ECs maintained their mitochondrial network. H- or H/RO-exposed ECs underwent changes, but mitochondrial fission was significantly less compared to that in ECs exposed to I/RP. I/RP-induced fission was partially inhibited by antioxidants, a NO synthase inhibitor, or an inhibitor of the fission protein dynamin-related protein 1 (Drp1) and was accompanied by Drp1 oligomerization and phosphorylation (Ser616). Hence, shear-induced NO, ROS (including mtROS), and Drp1 activation are responsible for mitochondrial fission in I/RP-exposed ECs, and excessive fission may be an underlying cause of EC dysfunction in postischemic hearts.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Traumatismo por Reperfusão/metabolismo , Superóxidos/metabolismo , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Hipóxia Celular , Células Cultivadas , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Forma das Organelas , Ácido Oxaloacético/farmacologia , Fosforilação , Multimerização Proteica , Resistência ao Cisalhamento , Estresse Mecânico
14.
J Pharmacol Exp Ther ; 329(1): 94-101, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19131585

RESUMO

Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (O(2)(*-)) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm(2), 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either N(G)-nitro-L-arginine methyl ester or N-acetyl-L-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges O(2)(*-), hydrogen peroxide (H(2)O(2)), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H(2)O(2) mediates the HO-1 induction. MitoSOX and 2',7'-dichlorofluorescin (DCF) fluorescence showed that mitochondrial O(2)(*-) levels and intracellular peroxides, respectively, are higher in sheared ECs compared with static controls and, in part, dependent on NO. SS-31 significantly inhibited both the shear-induced MitoSOX and DCF fluorescence signals. Either phosphatidylinositol 3-kinase or mitogen-activated protein kinase cascade inhibitors blocked the HO-1 induction. In conclusion, under shear, EC mitochondria-derived H(2)O(2) diffuses to the cytosol, where it initiates oxidative signaling leading to HO-1 up-regulation and maintenance of the atheroprotective EC status.


Assuntos
Células Endoteliais/metabolismo , Heme Oxigenase-1/biossíntese , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Western Blotting , Bovinos , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Espectrometria de Fluorescência
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