High-content single-cell drug screening with phosphospecific flow cytometry

Krutzik, Peter O.; Crane, Janelle; Clutter, Matthew R.; Nolan, Garry P.

doi:10.1038/nchembio.2007.59

Cited by 194 publications

(168 citation statements)

References 41 publications

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“…ERK phosphorylation was examined using a BD phosflow method as described. 25,26 Cell Depletion, Phagocytosis Inhibition, and Cell Adoptive Transfer. KCs were depleted by intravenous (i.v.)…”

Section: Methodsmentioning

confidence: 99%

Apoptotic cells attenuate fulminant hepatitis by priming Kupffer cells to produce interleukin-10 through membrane-bound TGF-β

Zhang

Yun

et al. 2010

Hepatology

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The liver, a unique tolerogenic organ, is regarded as the site to trap and destroy aging erythrocytes and activated T cells. However, to date, the mechanisms for why the liver is tolerogenic and whether liver Kupffer cells (KC) are critical phagocytes for apoptotic cells (AC) contributing to the liver immunosuppression remain unclear. Here we report that KC is the main phagocyte for AC in the liver. Contact of AC inhibits proinflammatory cytokine but enhances anti-inflammatory cytokine production of KC in response to lipopolysaccharide (LPS) stimulation. Membrane-bound transforming growth factor (TGF)-b on AC is responsible for the increased production of interleukin (IL)-10 in KC through extracellular signalregulated kinase (ERK) activation via the Smad3 pathway. Importantly, KC-derived IL-10 is critical for AC infusion-mediated protection of endotoxin-induced fulminant hepatitis through suppression of tumor necrosis factor (TNF)-a and nitric oxide (NO) production from KC and consequently attenuation of KC-mediated cytolysis of hepatocytes. Conclusion: AC can be preferentially phagocytosed by KC in the liver, leading to attenuation of fulminant hepatitis through IL-10-mediated suppression of KC-derived inflammatory TNF-a and NO production. These findings demonstrate that priming of KC by AC may contribute to maintain liver immunosuppression, providing a new mechanistic explanation for how immune homeostasis is maintained in the liver. (HEPATOLOGY 2011;53:306-316)

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Section: Methodsmentioning

confidence: 99%

Apoptotic cells attenuate fulminant hepatitis by priming Kupffer cells to produce interleukin-10 through membrane-bound TGF-β

Zhang

Yun

et al. 2010

Hepatology

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“…In signature-based approaches, a series of drug-induced molecular/phenotypic measurements are made in an experimental system. Collections of measurements from many small molecules form multivariate signatures that aim to fingerprint drugs based on their relative signature similarity (8)(9)(10)(11)(12)(13). In several landmark studies using Saccharomyces cerevisiae; gene expression compendia (8); and, later, barcoded loss of function/ORF libraries (9,10), large signatures were shown to characterize individual small molecule mechanisms of action effectively.…”

mentioning

confidence: 99%

Defining principles of combination drug mechanisms of action

Pritchard¹,

Bruno²,

Gilbert³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

154

137

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Combination chemotherapies have been a mainstay in the treatment of disseminated malignancies for almost 60 y, yet even successful regimens fail to cure many patients. Although their singledrug components are well studied, the mechanisms by which drugs work together in clinical combination regimens are poorly understood. Here, we combine RNAi-based functional signatures with complementary informatics tools to examine drug combinations. This approach seeks to bring to combination therapy what the knowledge of biochemical targets has brought to single-drug therapy and creates a statistical and experimental definition of "combination drug mechanisms of action." We show that certain synergistic drug combinations may act as a more potent version of a single drug. Conversely, unlike these highly synergistic combinations, most drugs average extant single-drug variations in therapeutic response. When combined to form multidrug regimens, averaging combinations form averaging regimens that homogenize genetic variation in mouse models of cancer and in clinical genomics datasets. We suggest surprisingly simple and predictable combination mechanisms of action that are independent of biochemical mechanism and have implications for biomarker discovery as well as for the development of regimens with defined genetic dependencies.chemotherapy | lymphoma | RNAi signature | systems biology C urrent rationales for the design of combination chemotherapy regimens were developed in the 1940s and 1950s (1-5) and, remarkably, were concurrent with the identification of DNA as the genetic material. The development of these regimens in the absence of any knowledge of cancer genomics was a remarkable achievement. However, although our knowledge of the genetic drivers of cancer and the mechanisms of drug action has increased dramatically over the past 30 y, this information has been difficult to adapt to clinical practice. As such, even very successful combination regimens often fail to cure many patients (6, 7). We hypothesize that part of this failure is due to the absence of mechanistic information about how drugs in regimens interact to promote combination effects (we term these effects "combination mechanisms of action"). We sought to address this gap by investigating specific hypotheses concerning the "mechanisms of action" of combination therapy.The classic term "drug mechanism of action" refers to the description of a specific biochemical event, which is often the activation or inhibition of an enzymatic effect. However, in recent years, "signature"-based prediction has provided a powerful new strategy for examining drug mechanism. In signature-based approaches, a series of drug-induced molecular/phenotypic measurements are made in an experimental system. Collections of measurements from many small molecules form multivariate signatures that aim to fingerprint drugs based on their relative signature similarity (8-13). In several landmark studies using Saccharomyces cerevisiae; gene expression compendia (8); and, later, barcoded loss o...

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“…Standard instruments nowadays have multiple laser colors as excitation light sources combined with automated preanalytics, multiple light detectors, or spectral analyzers to unravel the different color labels assigned to a plethora of surface or intracellular antigens or functional parameters. These instruments are, in combination with new assays and protocols (1,2), able to unravel new, hitherto unrecognized cell populations, cell signaling cascades, and so on.…”

mentioning

confidence: 99%

Beyond the flat world

Tárnok

2010

Cytometry Pt A

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High-content single-cell drug screening with phosphospecific flow cytometry

Cited by 194 publications

References 41 publications

Apoptotic cells attenuate fulminant hepatitis by priming Kupffer cells to produce interleukin-10 through membrane-bound TGF-β

Apoptotic cells attenuate fulminant hepatitis by priming Kupffer cells to produce interleukin-10 through membrane-bound TGF-β

Defining principles of combination drug mechanisms of action

Beyond the flat world

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