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Article: Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors

Conway, James R.W / Paul Timpson / Sean C. Warren

Methods. 2017 Sept. 01, v. 128

2017

Abstract: Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug ... ...

Abstract	Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.
Keywords	biosensors ; drugs ; fibrosis ; fluorescence ; hypoxia ; image analysis ; microscopy ; pH
Language	English
Dates of publication	2017-0901
Size	p. 78-94.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2017.04.014
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

Sean C. Warren / Anca Margineanu / Matilda Katan / Chris Dunsby / Paul M. W. French

International Journal of Molecular Sciences, Vol 16, Iss 7, Pp 14695-

2015 Volume 14716

Abstract: Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of ... ...

Abstract	Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation.
Keywords	time resolved fluorescence anisotropy imaging (TR-FAIM) ; FRET ; multiplexed imaging ; homo-FRET ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2015-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

George Chennell / Robin J. W. Willows / Sean C. Warren / David Carling / Paul M. W. French / Chris Dunsby / Alessandro Sardini

Sensors, Vol 16, Iss 8, p

2016 Volume 1312

Abstract: We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine ...

Abstract	We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.
Keywords	FRET ; FLIM ; AMPK ; spheroid ; 2-photon ; biosensor ; TCSPC ; 3D culture ; Technology (General) ; T1-995 ; Technology ; T ; Analytical chemistry ; QD71-142 ; Chemistry ; QD1-999 ; Science ; Q ; Chemical technology ; TP1-1185
Language	English
Publishing date	2016-08-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

Mark A Appaduray / Andrius Masedunskas / Nicole S Bryce / Christine A Lucas / Sean C Warren / Paul Timpson / Jeffrey H Stear / Peter W Gunning / Edna C Hardeman

PLoS ONE, Vol 11, Iss 12, p e

2016 Volume 0168203

Abstract: The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined ...

Abstract	The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.
Keywords	Medicine ; R ; Science ; Q
Subject code	612
Language	English
Publishing date	2016-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Three-dimensional organotypic matrices from alternative collagen sources as pre-clinical models for cell biology

James R. W. Conway / Claire Vennin / Aurélie S. Cazet / David Herrmann / Kendelle J. Murphy / Sean C. Warren / Lena Wullkopf / Alice Boulghourjian / Anaiis Zaratzian / Andrew M. Da Silva / Marina Pajic / Jennifer P. Morton / Thomas R. Cox / Paul Timpson

Scientific Reports, Vol 7, Iss 1, Pp 1-

2017 Volume 15

Abstract: Abstract Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of ...

Abstract	Abstract Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2017-12-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Removing physiological motion from intravital and clinical functional imaging data

Sean C Warren / Max Nobis / Astrid Magenau / Yousuf H Mohammed / David Herrmann / Imogen Moran / Claire Vennin / James RW Conway / Pauline Mélénec / Thomas R Cox / Yingxiao Wang / Jennifer P Morton / Heidi CE Welch / Douglas Strathdee / Kurt I Anderson / Tri Giang Phan / Michael S Roberts / Paul Timpson

eLife, Vol

2018 Volume 7

Abstract: Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for ... ...

Abstract	Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
Keywords	FLIM ; FRET ; motion correction ; intravital microscopy ; multiphoton ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2018-07-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

Sean C Warren / Anca Margineanu / Dominic Alibhai / Douglas J Kelly / Clifford Talbot / Yuriy Alexandrov / Ian Munro / Matilda Katan / Chris Dunsby / Paul M W French

PLoS ONE, Vol 8, Iss 8, p e

2013 Volume 70687

Abstract: Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting ... ...

Abstract	Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing ...
Keywords	Medicine ; R ; Science ; Q
Subject code	006 ; 530
Language	English
Publishing date	2013-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer

Alessia Floerchinger / Kendelle J. Murphy / Sharissa L. Latham / Sean C. Warren / Andrew T. McCulloch / Young-Kyung Lee / Janett Stoehr / Pauline Mélénec / Cris S. Guaman / Xanthe L. Metcalf / Victoria Lee / Anaiis Zaratzian / Andrew Da Silva / Michael Tayao / Sonia Rolo / Monica Phimmachanh / Ghazal Sultani / Laura McDonald / Susan M. Mason /

Nicola Ferrari / Lisa M. Ooms / Anna-Karin E. Johnsson / Heather J. Spence / Michael F. Olson / Laura M. Machesky / Owen J. Sansom / Jennifer P. Morton / Christina A. Mitchell / Michael S. Samuel / David R. Croucher / Heidi C.E. Welch / Karen Blyth / C. Elizabeth Caldon / David Herrmann / Kurt I. Anderson / Paul Timpson / Max Nobis

Cell Reports, Vol 36, Iss 11, Pp 109689- (2021)

Guidance using optical window intravital FRET imaging

2021

Abstract: Summary: Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) ...

Abstract	Summary: Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.
Keywords	intravital imaging ; small GTPases ; Rac1 ; FLIM ; FRET biosensors ; metastasis ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2021-09-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis

Monisha Samuel / Pamali Fonseka / Rahul Sanwlani / Lahiru Gangoda / Sing Ho Chee / Shivakumar Keerthikumar / Alex Spurling / Sai V. Chitti / Damien Zanker / Ching-Seng Ang / Ishara Atukorala / Taeyoung Kang / Sanjay Shahi / Akbar L. Marzan / Christina Nedeva / Claire Vennin / Morghan C. Lucas / Lesley Cheng / David Herrmann /

Nature Communications, Vol 12, Iss 1, Pp 1-

2021 Volume 16

Abstract: Dietary extracellular vesicles (EVs) could potentially be absorbed by the intestinal tract of the host and exert multiple phenotypic changes. Here, the authors isolate and characterize EVs from raw and commercial bovine milk and show orally administered ... ...

Abstract	Dietary extracellular vesicles (EVs) could potentially be absorbed by the intestinal tract of the host and exert multiple phenotypic changes. Here, the authors isolate and characterize EVs from raw and commercial bovine milk and show orally administered EVs to have a context specific role in promoting or suppressing primary tumor growth and metastasis in multiple mouse tumor models.
Keywords	Science ; Q
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Fluorescence lifetime readouts of Troponin-C-based calcium FRET sensors

Romain Laine / Daniel W Stuckey / Hugh Manning / Sean C Warren / Gordon Kennedy / David Carling / Chris Dunsby / Alessandro Sardini / Paul M W French

PLoS ONE, Vol 7, Iss 11, p e

a quantitative comparison of CFP and mTFP1 as donor fluorophores.

2012 Volume 49200

Abstract: We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, ...

Abstract	We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM ...
Keywords	Medicine ; R ; Science ; Q
Subject code	530 ; 500
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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